Human Cord Blood as a Source of Neural Tissue Repair of the Brain and Spinal Cord

ABSTRACT

The present invention relates to the use of umbilical cord blood cells from a donor or patient to provide neural cells which may be used in transplantation. The isolated cells according to the present invention may be used to effect autologous and allogeneic transplantation and repair of neural tissue, in particular, tissue of the brain and spinal cord and to treat neurodegenerative diseases of the brain and spinal cord.

FIELD OF THE INVENTION

The present invention relates to the use of human umbilical cord bloodand/or mononuclear cell fragment, thereof from a donor or patient toprovide neural cells for use in transplantation. The isolated cellsaccording to the present invention may be used to effect transplantationand repair of neural tissue, in particular, tissue of the brain andspinal cord and to treat neurodegenerative diseases and injury of thebrain and spinal cord.

BACKGROUND OF THE INVENTION

Neurobiologists believe that the neurons in the adult brain and spinalcord are impossible to rebuild once they are damaged. Thus, scienceprovided little hope to patients suffering from brain and spinal cordinjury or from neurodegenerative diseases such as Alzheimer's diseaseand Parkinson's disease, among a number of others. Parkinson's andAlzheimer's diseases are examples of neurodegenerative diseases whichare thought to be untreatable.

Parkinson's disease (PD), is a disorder of middle or late life, withvery gradual progression and a prolonged course. HARRISON'S PRINCIPLESOF INTERNAL MEDICINE, Vol. 2, 23d ed., Ed by Isselbacher, Braunwald,Wilson, Martin, Fauci and Kasper, McGraw-Hill Inc., New York City, 1994,pg. 2275. The most regularly observed changes in patients withParkinson's disease have been in the aggregates of melanin-containingnerve cells in the brainstem (substantia nigra, locus 20 coeruleus),where there are varying degrees of nerve cell loss with reactive gliosis(most pronounced in the substantia nigra) along with distinctiveeosinophilic intracytoplasmic inclusions. (Id. at 2276). In its fullydeveloped form, PD is easily recognized in patients, where stoopedposture, stiffness and slowness of movement, fixity of facialexpression, rhythmic tremor of the limbs, which subsides on activewilled movement or complete relaxation, are common features. Generally,accompanying the other characteristics of the fully developed disorderis the festinating gait, whereby the patient, progresses or walks withquick shuffling steps at an accelerating pace as if to catch up with thebody's center of gravity. (Id. at 2276).

The treatment of Parkinson's disease pharmacologically with levodopacombined with stereotactic surgery has only represented a partial cure,at best. (Id. at 2277). Underlying much of the treatment difficulty isdirected to the fact that none of these therapeutic measures has aneffect on the underlying disease process, which consists of neuronaldegeneration. Ultimately, a point seems to be reached where pharmacologycan no longer compensate for the loss of basal ganglia dopamine. (Id.).

Alzheimer's Disease (AD) is caused by a degenerative process in thepatient which is characterized by progressive loss of cells from thebasal forebrain, cerebral cortex and other brain areas. Acetylcholinetransmitting neurons and their target nerves are particularly affected.Senile plaques and neurofibrillary tangles are present. Pick's diseasehas a similar clinical picture to Alzheimer's disease but a somewhatslower clinical course and circumscribed atrophy, mainly affecting thefrontal and temporal lobes. One animal model for Alzheimer's disease andother dementias displays hereditary tendency toward the formation ofsuch plaques. It is thought that if a drug has an effect in the model,it also may be beneficial in at least some forms of Alzheimer's andPick's diseases. At present there are palliative treatments but no meansto restore function in Alzheimer's patients.

A group of related neuronal degenerative disorders is characterized byprogressive ataxia due to degeneration of the cerebellum, brainstem,spinal cord and peripheral nerves, and occasionally the basal ganglia.Many of these syndromes are hereditary; others occur sporadically. Thespinocerebellar degenerations are logically placed in three groups:predominantly spinal ataxias, cerebellar ataxias and multiple-systemdegenerations. To date there are no treatments. Friedrich's ataxia isthe prototypical spinal ataxia whose inheritance is autosomal recessive.The responsible gene has been found on Chromosome 9. Symptoms beginbetween ages of 5 and 15 with unsteady gait, followed by upper extremityataxia and dysarthria. Patients are flexic and lose large-fiber sensorymodalities (vibration and position sense). Two other diseases havesimilar symptoms: Bassen-Kornzweig syndrome (abeta-lipoproteinemia andvitamin E deficiency) and Refsom's disease (phytanic acid storagedisease). Cerebellar cortical degenerations generally occur between ages30 and 50. Clinically only signs of cerebellar dysfunction can bedetected, with pathologic changes have been reported. Similardegeneration may also be associated with chronic alcoholism. Inmultiple-system degenerations, ataxia occurs in young to middle adultlife in varying combinations with spasticity and extrapyramidal,sensory, lower motor neuron and autonomic dysfunction. In some families,there may also be optic atrophy, retinitis pigmentosa, opthalmoplegiaand dementia.

Another form of cerebellar degeneration is paraneoplastic cerebellardegeneration that occurs with certain cancers, such as oat cell lungcancer, breast cancer and ovarian cancer. In some cases, the ataxia mayprecede the discovery of the cancer by weeks to years. Purkinje cellsare permanently lost, resulting in ataxia. Even if the patient ispermanently cured of the cancer, their ability to function may beprofoundly disabled by the loss of Purkinje cells. There is no specifictreatment.

Strokes also result in neuronal degeneration and loss of functionalsynapses. Currently there is no repair, and only palliation andrehabilitation are undertaken.

Neurotransplantation has been used to explore the development of thecentral nervous system and for repair of diseased tissue in conditionssuch as Parkinson's and other neurodegenerative diseases. Theexperimental replacement of neurons by direct grafting of fetal tissueinto the brain has been accomplished in small numbers of patients inseveral research universities (including the University of SouthFlorida); but so far, the experimental grafting of human fetal neuronshas been limited by scarcity of appropriate tissue sources, logisticproblems, legal and ethical constraints, and poor survival of graftedneurons in the human host brain. One method replaces neurons by usingbone marrow stromal cells as stem cells for non-hematopoietic tissues.Marrow stromal cells can be isolated from other cells in marrow by theirtendency to adhere to tissue culture plastic. The cells have many of thecharacteristics of stem cells for tissues that can roughly be defined:as mesenchymal, because they can be differentiated in culture intoosteoblasts, chondrocytes. adipocytes, and even myoblasts. Thispopulation of bone marrow cells (BMSC) have also been used to preparedendritic cells, (K. Inaba, et al., J Experimental Med. 176: 1693-1702(1992)) which, as the name implies, have a morphology which might beconfused for neurons. Dendritic cells comprise a system ofantigen-presenting cells involved in the initiation of T cell responses.The specific growth factor, which stimulates production of dendriticcells, has been reported to be granulocyte/macrophage colony-stimulatingfactor 30 (GM-CSF). K. Inaba, et al., J Experimental Med. 176: 1693-1702(1992).

Work has recently been performed using stem cells obtained from bonemarrow to provide neural cells which can be used in neuronaltransplantation. See WO 99/56759. This patent represented theculmination of more than 130 years of work in the use of bone marrowstem cells for non-hematopoietic uses.

Several groups of investigators since 1990 have attempted to preparemore homogenous populations of stem cells from bone marrow. For example,U.S. Pat. No. 5,087,570, issued Feb. 11, 1992, discloses how to isolatehomogeneous mammalian hematopoietic stem cell compositions. Concentratedhematopoietic stem cell compositions are substantially free ofdifferentiated or dedicated hematopoietic cells. The desired cells areobtained by subtraction of other cells having particular markers. Theresulting composition may be used to provide for individual or groups ofhematopoietic lineages, to reconstitute stem cells of the host, and toidentify an assay for a wide variety of hematopoietic growth factors.

U.S. Pat. No. 5,633,426 issued May 27, 1997, is another example of thedifferentiation and production of hematopoietic cells. Chimericimmunocompromised mice are given human bone marrow of at least 4 weeksfrom the time of implantation. The bone marrow assumed the normalpopulation of bone marrow except for erythrocytes. These mice with humanbone marrow may be used to study the effect of various agents on theproliferation and differentiation of human hematopoietic cells.

U.S. Pat. No. 5,665,557, issued Sep. 9, 1997, relates to methods ofobtaining concentrated hematopoietic stem cells by separating out anenriched fraction of cells expressing the marker CDw 109. Methods ofobtaining compositions enriched in hematopoietic megakaryocyteprogenitor cells are also provided. Compositions enriched for stem cellsand populations of cells obtained therefrom are also provided by theinvention. Methods of use of the cells are also included.

U.S. Pat. No. 5,453,505 issued on Jun. 5, 1995, is yet another method ofdifferentiation. Primordial tissue is introduced into immunodeficienthosts, where the primordial tissue develops and differentiates. Thechimeric host allows for investigation of the processes and developmentof the xenogeneic tissue, testing for the effects of various agents onthe growth and differentiation of the tissue, as well as identificationof agents involved with the growth and differentiation.

U.S. Pat. No. 5,753,505 issued May 19, 1998, provides an isolatedcellular composition comprising greater than about 90% mammalian,non-tumor-derived, neuronal progenitor cells which express aneuron-specific marker and which can give rise to progeny which candifferentiate into neuronal cells. Also provided are methods of treatingneuronal disorders utilizing this cellular composition.

U.S. Pat. No. 5,759,793 issued Aug. 6, 1996, provides a method for boththe positive and negative selection of at least one mammalian cellpopulation from a mixture of cell populations utilizing a magneticallystabilized fluidized bed. One application of this method is theseparation and purification of hematopoietic cells. Target cellpopulations include human stem cells.

U.S. Pat. No. 5,789,148 issued Aug. 4, 1998, discloses a kit,composition and method for cell separation. The kit includes acentrifugable container and an organosilanized silica particle-basedcell separation suspension suitable for density gradient separation,containing a polylactam and sterilized by treatment with ionizingradiation. The composition includes a silanized silica particle-basedsuspension for cell separation which contains at least 0.05% of apolylactam. and preferably treated by ionizing radiation. Also disclosedis a method of isolating rare blood cells from a blood cell mixture.

Within the past several years, mesenchymal stem cells (MSCs) have beenexplored as vehicles for both cell therapy and gene therapy. The cellsare relatively easy to isolate from the small aspirates of bone marrowthat can be obtained under local anesthesia: they are also relativelyeasy to expand in culture and to transfect with exogenous genes.Prockop, D. J. Science 26: 71-74 (1997). Therefore, MSCs appear to haveseveral advantages over hematopoietic stem cells (HMCs) for use in genetherapy. The isolation of adequate numbers of HSCs requires largevolumes of marrow (1 liter or more), and the cells are difficult toexpand in culture. (Prockop, io D. J. (ibid.)).

There are several sources for bone marrow tissue, including thepatient's own bone marrow, that of blood relatives or others with MHCmatches and bone marrow banks. There are several patents that encompassthis source. U.S. Pat. No. 5,476,997 issued May 17, 1994, discloses amethod of producing human bone marrow equivalent. A human hematopoieticsystem is provided in an immunocompromised mammalian host, where thehematopoietic system is functional for extended periods of time. In thismethod, human fetal liver tissue and human fetal thymus are introducedinto a young immunocompromised mouse at a site supplied with a vascularsystem, whereby the fetal tissue results in formation of functionalhuman bone marrow tissue.

Human fetal tissue also represents a source of implantable neurons, butits use is quite controversial. U.S. Pat. No. 5,690,927 issued Nov. 25,1997, also utilizes human fetal tissue. Human fetal neuro-derived celllines are implanted into host tissues. The methods allow for treatmentof a variety of neurological disorders and other diseases.

U.S. Pat. No. 5,753,491, issued May 19, 1998, discloses methods fortreating a host by implanting genetically unrelated cells in the host.More particularly, the present invention provides human fetalneuro-derived cell lines, and methods of treating a host by implantationof these immortalized human fetal neuro-derived cells into the host. Onesource is the mouse, which is included in the U.S. Pat. No. 5,580,777issued Dec. 3, 1996. This patent features a method for the in vitroproduction of lines of immortalized neural precursor cells, includingcell lines having neuronal and/or glial cell characteristics, comprisesthe step of infecting neuroepithelium or neural crest cells with aretroviral vector carrying a member of the myc family of oncogenes.

U.S. Pat. No. 5,753,506 issued May 19, 1998, reveals an in vitroprocedure by which a homogeneous population of multipotential precursorcells from mammalian embryonic neuroepitheliurn (CNS stem cells) isexpanded up to 10 fold in culture while maintaining their multipotentialcapacity to differentiate into neurons, oligodendrocytes, andastrocytes. Chemical conditions are presented for expanding a largenumber of neurons from the stem cells. In addition, four factors—PDGF,CNTF, LIF, and T3—have been identified which, individually, generatesignificantly higher proportions of neurons, astrocytes, oroligodendrocytes. These procedures are intended to permit a large-scalepreparation of the mammalian CNS stem cells, neurons, astrocytes, andoligodendrocytes. These cells are proposed as an important tool for manycell- and gene-based therapies for neurological disorders. Anothersource of stem cells is that of primate embryonic stem cells. U.S. Pat.No. 5,843,780 issued Dec. 1, 1998, utilizes these stem cells. A purifiedpreparation of stem cells is disclosed. This preparation ischaracterized by the following cell surface markers: SSEA-I (−); SSEA-3(+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In oneembodiment, the cells of the preparation have normal karyotypes andcontinue to proliferate in an undifferentiated state after continuousculture for eleven months. The embryonic stem cells lines are alsodescribed as retaining the ability to form trophoblasts and todifferentiate into tissues derived from all three embryonic germ layers(endoderm, mesoderm and ectoderm). A method for isolating a primateembryonic stem cell line is also disclosed in the patent.

There is substantial evidence in both animal models and human patientsthat neural transplantation is a scientifically feasible and clinicallypromising approach to the treatment of neurodegenerative diseases andstroke as well as for repair of traumatic injuries to brain and spinalcord. Nevertheless, alternative cell sources and novel strategies fordifferentiation are needed to circumvent the numerous ethical andtechnical constraints that now limit the widespread use of neuraltransplantation. In short, there is a need for further development ofreadily available reliable sources of neural cells for transplantation.

The use of umbilical cord blood for use in hematopoietic reconstitutionhas been around since the work of Ende in the early 1970's. Becauseumbilical cord blood is rich in hematopoietic precursors, including stemcells, it represents a good source of cells for hematopoieticreconstitution. To date, however, little work has been done on usingpluripotential stem cells or related neural precursors which are foundin umbilical cord blood for neuronal transplantation perhaps because ofthe failure to realize the viable source of neuronal precursors whichcan be found in umbilical cord blood.

Human cord and placental blood provides a rich source of hematopoieticstem cells. On the basis of this finding, umbilical cord blood stemcells have been used to reconstitute hematopoiesis in children withmalignant and nonmalignant diseases after treatment with myeloablativedoses of chemoradiotherapy. Sirchia and Rebulla, 1999 Haematologica84:738-47. Early results show that a single cord blood sample providesenough hematopoietic stem cells to provide short- and long-termengraftment, and that the incidence and severity of graft-versus-hostdisease has been low even in HLA-mismatched transplants. These results,together with our previous discovery that bone marrow cells contain stemcells capable of differentiating into neurons and glia, led to thepresent invention which uses cord blood or mononuclear cell fractionsthereof to repair neuronal damage in brain and spinal cord.Sanchez-Ramos, et al. 1998. Movement Disorders 13(s2): 122 andSanchez-Ramos, et al., (2000) Exp. Neurol.

OBJECTS OF THE INVENTION

It is an object of the present invention to provide a novel source ofpluripotent stem and/or progenitor cells which can be readilydifferentiated into neuronal and glial cells to be used intransplantation in the brain and spinal cord of a patient and for thetreatment of neurodegenerative diseases.

It is an additional object of the invention to provide pharmaceuticalcompositions comprising effective amounts or concentrations of neuralcells for use in transplantation and methods for treatingneurodegenerative diseases, or brain or spinal cord injuries or damage.

It is another object of the invention to provide methods for isolatingand inducing differentiation of pluripotent stem and/or progenitor cellsinto neuronal and glial cells which can be used in transplantationprocedures or for the treatment of neurodegenerative diseases.

It is a further object to provide a method of treating neurodegenerativediseases and spinal cord/brain injury using neural and/or neuronaland/or glial cells derived from umbilical cord blood.

It is yet a further object of the invention to provide a method oftransplanting neural and/or neuronal and/or glial cells derived fromumbilical cord blood in order to repair damaged organs of a patient'snervous system such as the brain and spinal cord.

These and/or other objects of the invention may be readily gleaned fromthe description of the invention which follows.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected discovery that fresh orreconstituted umbilical cord blood or a mononuclear cellular fractionthereof is a novel source of cells which can be differentiated intoneural cells and/or neuronal tissue and used for neuronaltransplantation (autografting as well as allografting), thus obviatingthe need to use pooled neuronal fetal tissue or bone marrow tissue,which is often hard to obtain. Thus, the present invention may be usedfor autografting (cells from an individual are used in that sameindividual), allografting (cells from one person are used in anotherperson) and xenografting (transplantation from one species to another).In this aspect of the present invention, it has unexpectedly beendiscovered that umbilical cord blood from neonates or fetuses comprisescells which may be induced to become neurons in vitro and in vivo. Thesecells may be used in autologous or allogeneic transplantation (grafting)procedures to improve neurological deficit and to effect transplantationand repair of neural/neuronal tissue, in particular, tissue of the brainand spinal cord and to treat neurodegenerative diseases of the brain andspinal cord.

In one aspect according to the present invention, umbilical cord bloodderived neural cells are suitable for grafting into a patient's brain orspinal cord. These neural cells may be purified and/or incubated with adifferentiation agent by any one or more of the methods otherwisedescribed in the present specification or alternatively, these cells maybe obtained from crude mononuclear cell fractions of umbilical cordblood and used directly without further purification or differentiation.In other aspects, umbilical cord blood may be used without furtherpurification.

In another aspect of the present invention, there is presented a methodfor obtaining neural cells from umbilical cord blood, the methodcomprising the steps of obtaining umbilical cord blood, selectingumbilical cord pluripotential stem cells or progenitor cells which areneural precursor cells and incubating the umbilical cord stem cells orprogenitor cells with a differentiation agent to change the phenotype ofthe cells to produce a population of neural cells which are capable ofbeing transplanted. The steps of the method may also be changed suchthat all of the cells (for example, from an umbilical blood sample or amononuclear cell fraction thereof) are incubated with a differentiationagent prior to separation of the neural phenotype cells.

The method of the present invention may include the step of separatingthe pluripotential stem and progenitor cells from a population ofmononuclear cells obtained from umbilical cord blood using a magneticcell separator to separate out all cells which contain a CD marker, andthen expanding the cells which do not contain a marker in a growthmedium containing a differentiation agent such as retinoic acid, fetalneuronal cells or a growth factor such as BDNF, GDNF and NGF ormixtures, thereof, among numerous others. Preferably, a mixture ofretinoic acid and at least one growth factor, for example, nerve growthfactor, is used as the differentiation agent. The retinoic acid may be9-cis retinoic acid, all-transretinoic acid and mixtures thereof. Theseparation and incubation (differentiation) steps, may be interchanged.

Alternatively, an enriched cell population of pluripotent stem and/orprogenitor cells may be obtained from a population of mononuclear cellsobtained from umbilical cord blood by subjecting the mononuclearpopulation to an amount of an anti-proliferative agent (such as Ara-C[cytidine arabinoside] or methotrexate, among others) effective toeliminate all or substantially all proliferating cells and then exposingthe remaining non-proliferating cells to a mitogen such as epidermalgrowth factor or other mitogen (including other growth factors) toprovide a population of differentiated cells and quiescent cells(pluripotent stem or progenitor cells) which population is grown inculture medium such that the quiescent cells are concentrated in thecell population to greatly outnumber the differentiated cells. Thepluripotent stem and/or progenitor cells obtained may then be grown in acell medium containing a differentiation agent as generally describedabove in order to change the phenotype of the stem and/or progenitorcells to neuronal and/or glial cells which cells may be used intransplantation procedures directly without further purification.

The umbilical cord blood sample from which the pluripotent stem and/orprogenitor cells are obtained may be fresh umbilical cord blood,reconstituted cryopreserved umbilical cord blood or a fresh orreconstituted cryopreserved mononuclear fraction thereof.

Novel compositions according to the present invention comprise umbilicalcord blood or a mononuclear cellular fraction thereof, in combinationwith an effective amount of at least one neural cell differentiationagent. Neural cell differentiation agents for use in the presentinvention include for example, retinoic acid, fetal or mature neuronalcells including mesencephalic or striatal cells or a growth factor orcytokine such as brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF), glial growth factor (GFF) and nerve growthfactor (NGF) or mixtures, thereof. Additional differentiation agentsinclude, for example, growth factors such as fibroblast growth factor(FGF), transforming growth factors (TGF), ciliary neurotrophic factor(CNTF), bone-morphogenetic proteins (BMP), leukemia inhibitory factor(LIF), glial growth factor (GGF), tumor necrosis factors (TNF),interferon, insulin-like growth factors (IGF), colony stimulatingfactors (CSF), KIT receptor stem cell factor (KIT-SCF), interferon,triiodothyronine, thyroxine, erythropoietin, thrombopoietin, silencers,(including glial-cell missing, neuron restrictive silencer factor),antioxidants such as vitamin E (tocopherol) and vitamin E esters, amongothers including lipoic acid, SHC (SRC-homology-2-domain-containingtransforming protein), neuroproteins, proteoglycans, glycoproteins,neural adhesion molecules, and other cell-signalling molecules andmixtures, thereof.

Also presented is a cell line of pluripotent stem and/or progenitorcells produced by any one or more of the above-described methods suchthat the cells have the ability to migrate and localize to specificneuroanatomical regions where they differentiate into neuronal or glialcells typical of the region at the cite of transplantation and integrateinto the tissue in a characteristic tissue pattern. Pharmaceuticalcompositions utilizing these cells or other neural cells are also anaspect of the present invention.

The present invention also is directed to a kit for neuronaltransplantation comprising a flask with dehydrated culture medium and apluripotent stem and/or progenitor cells and/or other neural cells.

The present invention is also directed to a method for treating aneurodegenerative (preferably, transplanting in) a patient sufferingfrom such injury, a neurodegenerative disorder or neurological deficitan effective amount of neural and/or neuronal and/or glial cellsaccording to the present invention. Neurodegenerative disorders whichcan be treated using the method according to the present inventioninclude, for example, Parkinson's disease, Huntington's disease,multiple sclerosis (MS), Alzheimer's disease, Tay Sach's disease (betahexosaminidase deficiency), lysosomal storage disease, brain and/orspinal cord injury occurring due to ischemia, spinal cord and braindamage/injury, ataxia and alcoholism, among others, including a numberwhich are otherwise described herein.

The present invention is also directed to a method of treatingneurological damage in the brain or spinal cord which occurs as aconsequence of genetic defect, physical injury, environmental insult ordamage from a stroke, heart attack or cardiovascular disease (most oftendue to ischemia) in a patient, the method comprising administering(including transplanting), an effective number or amount of neural cellsobtained from umbilical cord blood to said patient, including directlyinto the affected tissue of the patient's brain or spinal cord.Administering cells according to the present invention to a patient andallowing the cells to migrate to the appropriate cite within the centralnervous system is another aspect of the present invention.

A method of obtaining neural and/or neuronal and/or glial cells forautologous transplantation from an individual's own umbilical cord bloodcomprises the steps of 1) harvesting mononuclear cells from fresh orcryopreserved umbilical cord blood or a cryopreserved mononuclearfraction of umbilical cord blood; 2) separating out the pluripotent stemcells and/or progenitor cells from the cord blood or mononuclearfraction; 3) incubating the stem cells and/or progenitor cells in amedium which includes an effective amount of a mitogen; and 4)incubating the stem and/or progenitor cells obtained from step 3 with adifferentiation agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is representative of results of gel studies indicating thepresence of mRNA from neuronal phenotypes.

FIG. 2 is representative of microscopic examination of immunostainedcultures of cells which are tested for immunoreactivity with antibodiesto neuronal markers. Certain of the figures evidence that the cells wereimmunoreactive with Mushashi-1 (FIG. 2A), β-tubulin III (FIG. 2B) andGFAP, a marker of astrocytes, (FIG. 2E).

FIGS. 3A, 3B and 3C show the results of neurological function recoveryin animals receiving a mononuclear fraction of human umbilical cordblood 1 day after MCAo as evidenced by adhesive removal, rotarod and NSStests.

FIGS. 4A, 4B and 4C show the results of neurological function recoveryin animals receiving a mononuclear fraction of human umbilical cordblood 7 days after MCAo as evidenced by adhesive removal, rotarod andNSS tests.

FIG. 5 shows the results of immunostaining of brain sections withMAB1281 and evidences that the highest concentrations of cord bloodcells migrate to the injured tissue.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are used throughout the specification todescribe the present invention.

The term “patient” is used throughout the specification to describe ananimal, preferably a human, to whom treatment, including prophylactictreatment, with the compositions according to the present invention, isprovided. For treatment of those infections, conditions or diseasestates which are specific for a specific animal such as a human patient,the term patient refers to that specific animal. The term “donor” isused to describe an individual (animal, including a human) who or whichdonates umbilical cord blood for use in a patient.

The term “umbilical cord blood” or “cord blood” is used throughout thespecification to refer to blood obtained from a neonate or fetus, mostpreferably a neonate and preferably refers to blood which is obtainedfrom the umbilical cord or the placenta of newborns. The use of cord orplacental blood as a source of mononuclear cells is advantageous becauseit can be obtained relatively easily and without trauma to the donor. Incontrast, the collection of bone marrow cells from a donor is atraumatic experience. Cord blood cells can be used for autologoustransplantation or allogenic transplantation, when and if needed. Cordblood is preferably obtained by direct drainage from the cord and/or byneedle aspiration from the delivered placenta at the root and atdistended veins.

The term “effective amount” is used throughout the specification todescribe concentrations or amounts of components such as differentiationagents, mitogens, neural and/or neuronal or glial cells, or other agentswhich are effective for producing an intended result includingdifferentiating stem and/or progenitor cells into neural, neuronaland/or glial cells, or treating a neurodegenerative disease or otherneurological condition including damage to the central nervous system ofa patient, such as a stroke, heart attack or accident victim or foreffecting a transplantation of those cells within the patient to betreated. Compositions according to the present invention may be used toeffect a transplantation of the neural cells within the composition toproduce a favorable change in the brain or spinal cord, or in thedisease or condition treated, whether that change is an improvement(such as stopping or reversing the degeneration of a disease orcondition, reducing a neurological deficit or improving a neurologicalresponse) or a complete cure of the disease or condition treated.

The term “neural cells” are cells having at least an indication ofneuronal or glial phenotype, such as staining for one or more neuronalor glial markers or which will differentiate into cells exhibitingneuronal or glial markers. Examples of neuronal markers which may beused to identify neuronal cells according to the present inventioninclude, for example, neuron-specific nuclear protein, tyrosinehydroxylase, microtubule associated protein, and calbindin, amongothers. The term neural cells also includes cells which are neuralprecursor cells, i.e., stem and/or progenitor cells which willdifferentiate into or become neural cells or cells which will ultimatelyexhibit neuronal or glial markers, such term including pluripotent stemand/or progenitor cells which ultimately differentiate into neuronaland/or glial cells. All of the above cells and their progeny areconstrued as neural cells for the purpose of the present invention.Neural stem cells are cells with the ability to proliferate, exhibitself-maintenance or renewal over the life-time of the organism and togenerate clonally related neural progeny. Neural stem cells give rise toneurons, astrocytes and oligodendrocytes during development and canreplace a number of neural cells in the adult brain. Neural stem cellsare neural cells for purposes of the present invention. The terms“neural cells” and “neuronal cells” are generally used interchangeablyin many aspects of the present invention. Preferred neural cells for usein certain aspects according to the present invention include thosecells which exhibit one or more of the neural/neuronal phenotypicmarkers such as Mushashi-1, Nestin, NeuN, class III β-tubulin, GFAP,NF-L, NF-M, microtubule associated protein (MAP2), S1100, CNPase,glypican (especially glypican 4), neuronal pentraxin II, neuronal PAS 1,neuronal growth associated protein 43, neurite outgrowth extensionprotein, vimentin, Hu, internexin, O4, myelin basic protein andpleiotrophin, among others.

The term “administration” or “administering” is used throughout thespecification to describe the process by which neural cells according tothe present invention are delivered to a patient for treatment purposes.Neural cells may be administered a number of ways including parenteral(such term referring to intravenous and intraarterial as well as otherappropriate parenteral routes), intrathecal, intraventricular,intraparenchymal (including into the spinal cord, brainstem or motorcortex), intracisternal, intracranial, intrastriatal, and intranigral,among others which term allows neural cells to migrate to the cite whereneeded. Neural cells may be administered in the form of whole cord bloodor a fraction thereof (such term including a mononuclear fractionthereof or a fraction of neural cells, including a high concentration ofneural cells). The compositions according to the present invention maybe used without treatment with a differentiation agent (“untreated”,i.e., without further treatment in order to promote differentiation ofcells within the umbilical cord blood sample) or after treatment(“treated”) with a differentiation agent or other agent which causescertain pluripotential stem and/or progenitor cells within the cordblood sample to differentiate into cells exhibiting neuronal and/orglial phenotype. Administration will often depend upon the disease orcondition treated and may preferably be via a parenteral route, forexample, intravenously, by administration into the cerebral spinal fluidor by direct administration into the affected tissue in the brain. Forexample, in the case of Alzheimer's disease, Huntington's disease andParkinson's disease, the preferred route of administration will be atransplant directly into the striatum (caudate cutamen) or directly intothe substantia nigra (Parkinson's disease). In the case of amyotrophiclateral sclerosis (Lou Gehrig's disease) and multiple sclerosis, thepreferred administration is through the cerebrospinal fluid. In the caseof lysosomal storage disease, the preferred route of administration isvia an intravenous route or through the cerebrospinal fluid. In the caseof stroke, the preferred route of administration will depend upon wherethe stroke is, but will often be directly into the affected tissue(which may be readily determined using MRI or other imaging techniques).

Each of these conditions, however, may be readily treated using otherroutes of administration including, for example, an intravenous orintraarterial administration of whole umbilical cord blood or amononuclear cell fraction thereof to treat a condition or disease state.In the case of such treatment, however, and in particular, the treatmentof amyotrophic lateral sclerosis (Lou Gehrig's disease), treatment ofthe patient using parenteral (in particular, intravenous orintraarterial) administration of whole umbilical cord blood or amononuclear cellular fraction thereof or other routes of administrationwill be performed preferably in the absence of radiation or othertreatment such as chemotherapy (which are often used to eliminate bonemarrow cells or other tissue in the patient in order to impair, destroyand replace hematopoietic cells) before, during or after administrationof the umbilical cord blood or mononuclear cell fraction, thereof.

The terms “grafting” and “transplanting” and “graft” and“transplantation” are used throughout the specification synonymously todescribe the process by which neural and/or neuronal cells according tothe present invention are delivered to the site within the nervoussystem where the cells are intended to exhibit a favorable effect, suchas repairing damage to a patient's central nervous system, treating aneurodegenerative disease or treating the effects of nerve damage causedby stroke, cardiovascular disease, a heart attack or physical injury ortrauma or genetic damage or environmental insult to the brain and/orspinal cord, caused by, for example, an accident or other activity.Neural cells for use in the present invention may also be delivered in aremote area of the body by any mode of administration as describedabove, relying on cellular migration to the appropriate area in thecentral nervous system to effect transplantation.

The term “essentially” is used to describe a population of cells or amethod which is at least 95+% effective, more preferably at least about98% effective and even more preferably at least 99% effective. Thus, amethod which “essentially” eliminates a given cell population,eliminates at least about 95+% of the targeted cell population, mostpreferably at least about 99% of the cell population. Neural cellsaccording to the present invention, in certain preferred embodiments,are essentially free of hematopoietic cells (i.e., the CD34+ cellularcomponent of the mononuclear cell fragment).

The term “non-tumorigenic” refers to the fact that the cells do not giverise to a neoplasm or tumor. Stem and/or progenitor cells for use in thepresent invention are generally free from neoplasia and cancer.

The term “cell medium” or “cell media” is used to describe a cellulargrowth medium in which mononuclear cells and/or neural cells are grown.Cellular media are well known in the art and comprise at least a minimumessential medium plus optional agents such as growth factors, glucose,non-essential amino acids, insulin, transferrin and other agents wellknown in the art. In certain preferred embodiments at least onedifferentiation agent is added to the cell media in which a mononuclearcell fraction is grown in order to promote differentiation of certaincells within the mononuclear fraction into neural cells.

In a preferred aspect of the present invention, mononuclear cells grownin standard cellular media (preferably, at least a minimum essentialmedium supplemented with non-essential amino acids, glutamine,mercaptoethanol and fetal bovine serum (FBS)) are grown in a “neuralproliferation medium” (i.e., a medium which efficiently grows neuralcells) followed by growth in a “differentiation medium”, generally,which is similar to the neural proliferation medium with the exceptionthat specific nerve differentiation agents are added to the medium andin certain cases, other growth factors are limited or removed). Aparticularly preferred neural proliferation medium is a media whichcontains DMEM/F12 1:1 cell media, supplemented with 0.6% glucose,insulin (25 μg/ml), transferrin (100 μg/ml), progesterone 20 nM,putrescine (60 μM, selenium chloride 30 nM, glutamine 2 mM, sodiumbicarbonate 3 mM, HEPES 5 mM, heparin 2 μg/ml and EGF 20 nm/ml, bFGF 20ng/ml. One of ordinary skill will readily recognize that any number ofcellular media may be used to grow mononuclear cell fractions ofumbilical cord blood or to provide appropriate neural proliferationmedia and/or differentiation media.

The term “separation” is used throughout the specification to describethe process by which pluripotent stem and/or progenitor cells areisolated from a mononuclear cell sample or a sample which contains cellsother than the desirable stem and/or progenitor cells, for example,umbilical cord blood or other fragment.

The term “mitogen” is used throughout the specification to describe anagent which is added to non-proliferating cells obtained from amononuclear cell sample in order to produce differentiated cells andquiescent cells (pluripotent stem and/or progenitor cells). A mitogen isa transforming agent which induces mitosis in certain cells other thanpluripotent stem and/or progenitor cells obtained from umbilical cordblood. Preferred mitogens for use in the present invention includeepidermal growth factor (EGF), among other agents such as the lesspreferred pokeweed mitogen, which also may be used to induce mitosis.Mitogens are also any one or a combination of a variety of growthfactors which have been shown to exert mitogenic actions on neural andmesenchymal precursors. These growth factors are: Epidermal GrowthFactor (EGF) family ligands (EGF, Transforming Growth Factor α,amphiregulin, betacellulin, heparin-binding EGF and Heregulin), basicFibroblastic growth factors (bFGF) and other members of its super-family(FGF1, FGF4), members of Platelet-Derived Growth Factor family (PDGF AA,AB, BB), Interleukins, and members of the Transforming Growth Factor βsuperfamily.

The term “antiproliferative agent” is sued throughout the specificationto describe an agent which will prevent the proliferation of cellsduring methods according to the present invention which enrichpluripotent stem and/or progenitor cells. Exemplary antiproliferativeagents include, for example, Ara-C, methotrexate and otherantiproliferative agents. Preferred antiproliferative agents are thoseagents which limit or prevent the growth of proliferating cells withinan umbilical cord blood sample or mononuclear cell fraction thereof sothat quiescent stem and/or progenitor cells may be enriched.

The term “differentiation agent” or “neural differentiation agent” isused throughout the specification to describe agents which may be addedto cell culture (which term includes any cell culture medium which maybe used to grow neural cells according to the present invention)containing pluripotent stem and/or progenitor cells which will inducethe cells to a neuronal or glial phenotype. Preferred differentiationagents for use in the present invention include, for example,antioxidants, including retinoic acid, fetal or mature neuronal cellsincluding mesencephalic or striatal cells or a growth factor or cytokinesuch as brain derived neurotrophic factor (BDNF), glial derivedneurotrophic factor (GDNF) and nerve growth factor (NGF) or mixtures,thereof. Additional differentiation agents include, for example, growthfactors such as fibroblast growth factor (FGF), transforming growthfactors (TGF), ciliary neurotrophic factor (CNTF), bone-morphogeneticproteins (BMP), leukemia inhibitory factor (LIF), glial growth factor(GGF), tumor necrosis factors (TNF), interferon, insulin-like growthfactors (IGF), colony stimulating factors (CSF), KIT receptor stem cellfactor (KIT-SCF), interferon, triiodothyronine, thyroxine,erythropoietin, thrombopoietin, silencers, (including glial-cellmissing, neuron restrictive silencer factor), SHC(SRC-homology-2-domain-containing transforming protein), neuroproteins,proteoglycans, glycoproteins, neural adhesion molecules, and othercell-signalling molecules and mixtures, thereof. Differentiation agentswhich can be used in the present invention are detailed in“Marrow-mindedness: a perspective on neuropoiesis”, by Bjorn Scheffler,et al., TINS, 22, pp. 348-356 (1999), which is incorporated by referenceherein.

The term “neurodegenerative disease” is used throughout thespecification to describe a disease which is caused by damage to thecentral nervous system and which damage can be reduced and/or alleviatedthrough transplantation of neural cells according to the presentinvention to damaged areas of the brain and/or spinal cord of thepatient. Exemplary neurodegenerative diseases which may be treated usingthe neural cells and methods according to the present invention includefor example, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, RettSyndrome, lysosomal storage disease (“white matter disease” orglial/demyelination disease, as described, for example by Folkerth, J.Neuropath. Exp. Neuro., 58, 9, September, 1999), including Sanfilippo,Tay Sachs disease (beta hexosaminidase deficiency), other geneticdiseases, multiple sclerosis, brain injury or trauma caused by ischemia,accidents, environmental insult, etc., spinal cord damage, ataxia andalcoholism. In addition, the present invention may be used to reduceand/or eliminate the effects on the central nervous system of a strokeor a heart attack in a patient, which is otherwise caused by lack ofblood flow or ischemia to a site in the brain of said patient or whichhas occurred from physical injury to the brain and/or spinal cord. Theterm neurodegenerative diseases also includes neurodevelopmentaldisorders including for example, autism and related neurologicaldiseases such as schizophrenia, among numerous others.

Selecting for umbilical cord pluripotential stem and/or progenitor cellsaccording to the present invention can be done in a number of ways. Forexample, the cells may be selected using, for example a magnetic cellseparator (FACS) or other system which removes all cells which contain aCD marker and then the remaining cells may be expanded in growth mediumor differentiated in growth medium which includes a differentiationagent. Alternatively, an enriched population of stem and/or progenitorcells may be obtained from a sample of mononuclear cells by subjectingthe cells to an agent such as Ara-C or other anti-proliferative agentsuch as methotrexate, which causes the death of proliferating cellswithin a sample (the stem and/or progenitor cells are non-proliferatingand are unaffected by the agent). The remaining cells may then be grownin a cell culture medium which contains a mitogen to produce apopulation of differentiated and quiescent cells, which cell populationmay be further grown to concentrate the quiescent cells to the effectiveexclusion of the differentiated cells (the quiescent cells in the finalcell medium will greatly outnumber the original differentiated cellswhich do not grow in the medium). The quiescent cells may then beinduced to adopt a number of different neural phenotypes, which cellsmay be used directly in transplantation.

Additional in vitro differentiation techniques can be adapted throughthe use of various cell growth factors and co-culturing techniques knownin the art. Besides co-culturing with fetal mesencephalic or striatalcells, a variety of other cells can be used, including but not limitedto accessory cells, and cells from other portions of the fetal andmature central nervous system.

The term “gene therapy” is used throughout the specification to describethe transfer and stable insertion of new genetic information into cellsfor the therapeutic treatment of diseases or disorders. The foreign geneis transferred into a cell that proliferates to spread the new genethroughout the cell population. Thus, stem cells, or pluripotentprogenitor cells according to the present invention either prior todifferentiation or preferably, after differentiation to a neural cellphenotype, are the target of gene transfer, since they are proliferativecells that produce various progeny lineages which will potentiallyexpress the foreign gene.

The following written description provides exemplary methodology andguidance for carrying out many of the varying aspects of the presentinvention.

General Methods

Standard molecular biology techniques known in the art and notspecifically described are generally followed as in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory,New York (1989, 1992), and in Ausubel et al., Current Protocols InMolecular Biology, John Wiley and Sons, Baltimore, Md. (1989).Polymerase chain reaction (PCR) is carried out generally as in PCRProtocols: A Guide To Methods and Applications, Academic Press, SanDiego, Calif. (1990). Reactions and manipulations involving othernucleic acid techniques, unless stated otherwise, are performed asgenerally described in Sambrook, et al., 1989, Molecular Cloning: aLaboratory Manual, Cold Springs Harbor Laboratory Press, and methodologyas set forth in U.S. Pat. Nos. 4,666,828; 4,683,202, 4,801,531;5,192,659; and 5,272,057 and incorporated herein by reference. In-situPCR in combination with Flow Cytometry can be used for detection ofcells containing specific DNA and mRNA sequences (see, for example,Testoni et al. Blood 87:3822 (1996)).

Standard methods in immunology known in the art and not specificallydescribed are generally followed as in Stites et al. (eds), BASIC ANDCLINICAL IMMUNOLOGY, 8'th Ed., Appleton & Lange, Norwalk, Conn. (1994);and Mishell and Shigi (eds), Selected Methods In Cellular Immunology,W.H. Freeman and Co., New York (1980).

Immunoassays

In general, immunoassays are employed to assess a specimen such as forcell surface markers or the like. Immunocytochemical assays are wellknown to those skilled in the art. Both polyclonal and monoclonalantibodies can be used in the assays. Where appropriate otherimmunoassays, such as enzyme-linked immunosorbent assays (ELISAs) andradioimmunoassays (RIA), can be used as are known to those in the art.Available example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752;3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074;3,984,533; 3,996,345; 4,034,074; 4,098,876; 2o 4,879,219; 5,011,771 and5,281,521 as well as Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Springs Harbor, N.Y. (1989). Numerous other references alsomay be relied on for these teachings.

Antibody Production

Antibodies may be monoclonal, polyclonal or recombinant. Conveniently,the antibodies may be prepared against the immunogen or immunogenicportion thereof, for example, a synthetic peptide based on the sequence,or prepared recombinantly by cloning techniques or the natural geneproduct and/or portions thereof may be isolated and used as theimmunogen. Immunogens can be used to produce antibodies by standardantibody production technology well known to those skilled in the art asdescribed generally in Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Springs Harbor, N.Y. (1988) andBorrebaeck, Antibody Engineering—A Practical Guide by W. H. Freeman andCo. (1992). Antibody fragments may also be prepared from the antibodiesand include Fab and F(ab′)2 by methods known to those skilled in theart. For producing polyclonal antibodies a host, such as a rabbit orgoat, is immunized with the immunogen or immunogenic fragment, generallywith an adjuvant and, if necessary, coupled to a carrier; antibodies tothe immunogen are collected from the serum. Further, the polyclonalantibody can be absorbed such that it is monospecific. That is, theserum can be exposed to related immunogens so that cross-reactiveantibodies are removed from the serum rendering it monospecific.

For producing monoclonal antibodies, an appropriate donor ishyperimmunized with the immunogen, generally a mouse, and splenicantibody-producing cells are isolated. These cells are fused to immortalcells, such as myeloma cells, to provide a fused cell hybrid that isimmortal and secretes the required antibody. The cells are thencultured, and the monoclonal antibodies harvested from the culturemedia.

For producing recombinant antibodies, messenger RNA fromantibody-producing B-lymphocytes of animals or hybridoma isreverse-transcribed to obtain complementary DNAs (cDNAs). Antibody cDNA,which can be full or partial length, is amplified and cloned into aphage or a plasmid. The cDNA can be a partial length of heavy and lightchain cDNA, separated or connected by a linker. The antibody, orantibody fragment, is expressed using a suitable expression system.Antibody cDNA can also be obtained by screening pertinent expressionlibraries. The antibody can be bound to a solid support substrate orconjugated with a detectable moiety or be both bound and conjugated asis well known in the art. (For a general discussion of conjugation offluorescent or enzymatic moieties see Johnstone & Thorpe,Immunochemistry in Practice, Blackwell Scientific Publications, Oxford,1982). The binding of antibodies to a solid support substrate is alsowell known in the art. (see for a general discussion Harlow & Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPublications, New York, 1988 and Borrebaeck, Antibody Engineering—APractical Guide, W.H. Freeman and Co., 1992). The detectable moietiescontemplated with the present invention can include, but are not limitedto, fluorescent, metallic, enzymatic and radioactive markers. Examplesinclude biotin, gold, ferritin. alkaline phosphates, galactosidase,peroxidase, urease, fluorescein, rhodamine, tritium, ¹⁴C, iodination andgreen fluorescent protein.

Gene Therapy

Gene therapy as used herein refers to the transfer of genetic material(e.g., DNA or RNA) of interest into a host to treat or prevent a geneticor acquired disease or condition. The genetic material of interestencodes a product (e.g., a protein. polypeptide. and peptide, functionalRNA, antisense) whose in vivo production is desired. For example, thegenetic material of interest encodes a hormone, receptor, enzyme,polypeptide or peptide of therapeutic value. Alternatively, the geneticmaterial of interest encodes a suicide gene. For a review see “GeneTherapy” in Advances In Pharmacology, Academic Press, San Diego, Calif.,1997.

Administration of Cells for Transplantation

The cells of the present invention are administered and dosed inaccordance with good medical practice, taking into account the clinicalcondition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement, including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

In the method of the present invention, the cells of the presentinvention can be administered in various ways as would be appropriate toimplant in the central nervous system, including but not limited toparenteral, including intravenous and intraarterial administration,intrathecal administration, intraventricular administration,intraparenchymal, intracranial, intracisternal, intrastriatal, andintranigral administration. In addition, all of these routes ofadministration may be used to effect transplantation of neural cells inthe present invention.

Methods of treating a patient for a neurodegenerative disease or brainand/or spinal cord damage caused by, for example, physical injury or byischemia caused by, a stroke, heart attack or cardiovascular diseasecomprise administering neural cells to said patient in an amountsufficient to effect a neuronal transplantation. One of ordinary skillmay readily recognize that one may use treated (i.e., cells exposed toat least one differentiation agent) or untreated neural cells for suchmethods, including fresh umbilical cord blood or a mononuclear fractionthereof.

Pharmaceutical compositions comprising effective amounts of treatedneural cells are also contemplated by the present invention. Thesecompositions comprise an effective number of treated neural and/orneuronal and/or glial cells, optionally in combination with apharmaceutically acceptable carrier, additive or excipient. In certainaspects of the present invention, cells are administered to the patientin need of a transplant in sterile saline. In other aspects of thepresent invention, the cells are administered in Hanks Balanced SaltSolution (HBSS) or Isolyte S, pH 7.4. Other approaches may also be used,including the use of serum free cellular media. Such compositions,therefore, comprise effective amounts or numbers of treated neural cellsin sterile saline. These may be obtained directly by using fresh orcryopreserved umbilical cord blood or alternatively, by separating outthe mononuclear cells (MNC) from the whole blood, using density gradientseparation methods, among others, which are well known in the art (onesuch approach is presented herein). The isolated MNC may be useddirectly for administration/transplantation or may be treated with atleast one differentiation agent and used without further purification orisolation of neural cells, or alternatively, after treatment with atleast one differentiation agent, the neural cells may be isolated andused. Intravenous or intraarterial administration of the cells insterile saline to the patient may be preferred in certain indications,whereas direct administration at the site of or in proximity to thediseased and/or damaged tissue may be preferred in other indications.

Pharmaceutical compositions according to the present inventionpreferably comprise an effective number within the range of about1.0×10⁴ mononuclear cells to about 5.0×10⁷ mononuclear cells, morepreferably about 1×10⁵ to about 9×10⁶ mononuclear cells, even morepreferably about 1×10⁶ to about 8×10⁶ cells generally in solution,optionally in combination with a pharmaceutically acceptable carrier,additive or excipient. Effective numbers of neural cells, either withina sample of mononuclear cells or as concentrated or isolated neuralcells, may range from as few as several hundred or fewer to severalmillion or more, preferably at least about one thousand cells withinthis range. In aspects of the present invention whereby the cells areinjected in proximity to the brain or spinal cord tissue to be treated,the number of cells may be reduced as compared to aspects of the presentinvention which rely on parenteral administration (including intravenousand/or intraarterial administration).

In using compositions according to the present invention, fresh orcryopreserved umbilical cord blood, a mononuclear fraction thereof, orfractions wherein neural cells are isolated and/or concentrated (usingFACS or other separation methods for isolating neural cells from apopulation of mononuclear cells) may be used without treatment with adifferentiation agent or with an effective amount of a differentiationagent prior to being used in a neuronal transplant. In one preferredaspect of the present invention, a mononuclear fraction of cord blood isexposed to effective amounts of at least one differentiation agent (incell media) for a period to effect differentiation of cord stem cellsinto cells which express a neuronal and/or glial phenotype and aftersuch period, these cells are then used to effect a transplant in apatient.

In aspects of the invention in which cord blood stem cells aredifferentiated, the use of standard media which has been supplementedwith at least one or more differentiation agent is preferred. Acombination of retinoic acid and nerve growth factor (NGF) in effectiveamounts in certain aspects of the present invention as thedifferentiation agent is preferred. In certain preferred aspects of thepresent invention, neural cells are prepared from cord blood stems cellsgrowing in standard growth media in a two step approach using neuralproliferation media followed by a differentiation media. In this aspectof the present invention, cells grown in standard cellular media(preferably, at least a minimum essential medium supplemented withnon-essential amino acids, glutamine, mercaptoethanol and fetal bovineserum (FBS)) are initially grown in a “neural proliferation medium”(i.e., a medium which efficiently grows neural cells) followed by growthin a “differentiation medium”) (generally, similar to the neuralproliferation medium with the exception that specific nervedifferentiation agents are added to the medium and in certain cases,other growth factors are limited or removed). A preferred neuralproliferation medium is a media which contains DMEM/F12 1:1 cell media,supplemented with 0.6% glucose, insulin (25 μg/ml), transferrin (100μg/ml), progesterone 20 nM, putrescine (60 μM, selenium chloride 30 nM,glutamine 2 mM, sodium bicarbonate 3 mM, HEPES 5 mM, heparin 2 μg/ml andEGF 20 nm/ml, bFGF 20 ng/ml. One of ordinary skill will readilyrecognize that any number of cellular media may be used to provideappropriate neural proliferation medium and/or differentiation medium.

The following examples are provided to illustrate or exemplify certainpreferred embodiments of the present invention illustrative of thepresent invention but are not intended in any way to limit the presentinvention.

EXAMPLES

Preparation of Cellular Samples

Cryopreserved or fresh umbilical cord blood (from human or rat umbilicalcord that remains attached to placenta after delivery) is harvested andprocessed by Ficoll centrifugation. This results in nearly 100% recoveryof mononuclear cells which can be a) grafted directly into a region ofinjured brain (e.g. in a rat stroke model or model of neurodegenerativedisease or trauma model) b) processed into sub-populations based onsurface markers or c) cryopreserved for later use. Initial experimentswith umbilical cord blood utilize all of the mononuclear cellscollected, without separation of CD34+ cellular components(hematopoietic stem cells). Other experiments utilize cord blood that isdepleted of CD34+ cells as described below. Approximately 100,000 to90,000,000 (1×10⁵ to about 9×10⁷, preferably at least about 1×10⁶) cordblood cells are injected into the hemisphere rendered ischemic byacutely obstructing blood flow to cerebral cortex. Assessment ofrecovery of limb function in the rat model of stroke is performed at 2and 4 and 8 weeks after grafting.

Preparation of Cord Blood Devoid of Hematopoietic Stem Cells (CD34+)

Using a magnetic cell sorting kit (Milteny Biotec, Inc, Auburn Tx), cordcells are labeled with CD34+ microbeads which marks cells that expresshematopoietic stem cell antigen (CD34 in human samples). The cord bloodcells are passed through an MS+ column for selection of CD34+ cells. Twohundred μL of CD34+ Multi-sort MicroBeads is added per 10⁸ total cells,mixed and incubated for 15 min at 6-12° C. Cells are washed by adding5-10× the labeling volume of buffer, centrifuged for 10 min at 200×g andsupernatant is removed. The cell pellet is resuspended in 500 μL buffer.The MS+/RS+ column is washed with 500 μL of buffer. The cell suspensionis applied to the column and the “negative” cells passed through. Thenegative cells contain all cells in the cord blood except thehematopoietic CD34+ cells. The column is then rinsed with 500 μL ofbuffer three times. These washing are added to the “negative” cellfraction, centrifuged for 10 min at 220×g and the supernatant removed.The cell pellet is resuspended in 500 μL of buffer and diluted 1:1 withDulbecco's Minimal Essential Media (DMEM, GIBCO/BRL) and 10% fetalbovine serum (FBS), centrifuged through a density gradient (Ficoll-PaquePlus, 1.077 g/ml, Pharmacia) for 30 min at 1,000×g. The supernatant andinterface are combined and diluted to approximately 20 ml with growthmedium and plated in polyethylene-imine coated plastic flasks.

Defining the Optimal Medium for Generating Cord Blood Clones

The cord blood is suspended in serum-free medium composed of a 1:1mixture of Dulbecco's Minimal Essential Media (DMEM) and F12 nutrient(Life Technologies-BRL). Other samples of cord blood material aresuspended in DMEM+10% Fetal Bovine Serum (FBS). The defined culturemedium is composed of DMEM/F12 (1:1) including glucose, glutamine,sodium bicarbonate and HEPES buffer plus a defined hormone and saltmixture (see Daadi and Weiss, 1999). To identify the optimal celldensity for cell survival and growth, cells are plated at densitiesvarying from 50,000 to 3×10⁶ cell/ml in Corning T75 culture flasks inthe defined media together with a specific mitogenic growth factor(s)(see below for the mitogens used). Cell survival and proliferation aremonitored very closely by examining culture flasks, noting and countingclones that arise daily. After a fixed culture period of 10 days thetotal number of clones are counted in both serum free and serumsupplemented cultures and compared. This gave us the number of precursorcells able to generate clones and rate of proliferation. Then theseclones are harvested, dissociated and the total viable cells counted andreseeded for a second passage and so on for the future passages. Thislast count allowed determination of the rate of proliferation and thesize of clones generated under each condition.

Caveats: From our experience with neural stem cells, we have found thatexposure of cells to serum induces differentiation and inhibitsproliferation of neural lineages. Lower cell density also does not favorcell proliferation. Therefore, it is likely that we will observevariability in the rate of cell growth depending on the presence andabsence of serum, the cell density and mitogen used. The mediacomposition may not be optimal for cell survival, proliferation andenrichment for a neural cell population. We also see differences inmorphologies and antigens expressed by the cells under these twoseparate conditions. Therefore, before each passage we identify thetotal number of clones and cells and also the proportion of neurons,astrocytes and oligodendrocytes as well as hematopoietic cell lineages.These data will guide us to constantly improve our medium formula bytrying new media components and, if necessary, a very low percentage ofBovine Serum Albumin (BSA) or neural stem cell conditioned media.

Identification of Sub-Populations of Cord Blood Cells Responsive toSpecific Mitogens and Which Express Specific Neural Markers

The cord blood suspension is plated at an optimal cell density in comingT75 culture flasks in the optimal medium (as described above). Thismedium is supplemented with 10 to 20 ng/ml of one or a combination of avariety of growth factors that have been shown to exert mitogenicactions on neural and mesenchymal precursors. These growth factors are:Epidermal Growth Factor (EGF) family ligands (EGF, Transforming GrowthFactor α, amphiregulin, betacellulin, heparin-binding EGF andHeregulin), basic Fibroblastic growth factors (bFGF) and other membersof its super-family (FGF1, FGF4), members of Platelet-Derived GrowthFactor family (PDGF AA, AB, BB), Interleukins, and members of theTransforming Growth Factor β superfamily. After a period of 10 to 15days in vitro, the cells are harvested and then reseeded in fresh mediumcontaining growth factor(s). To identify their immature nature, some ofthese cells are plated on poly-L-ornithine-coated glass coverslips in24-well Nunclon culture dishes. After, a period of 30 minutes to 1 hourthese cells are fixed with 4% paraformaldehyde and stained with avariety of markers for immature cells such as Nestin, vimentin, the CDmarkers CD34 (marker of hematopoietic stem cell) and CD33 and dendriticcell markers. The rest of the cells are reseeded in growth medium(medium containing mitogens) for the next passage. For each growthfactor used cell survival and proliferation is closely monitored andclones that arise every day are counted and the total number of clonesformed after a fixed period is determined. We also determine theproportion of cell types generated under each mitogen, as describedbelow.

Different rates of cell proliferation and/or proportion of cell typesare generated depending on the growth factor used and its concentration.Neuronal differentiation efficacy and the number of passages able to becarried out under each mitogen is determined. Studies have shown thatthe combination of EGF and FGF is required to isolate and propagatehuman neural stem cell. Therefore we test combinations of these growthfactors and potentiated the mitogenic action by adding a specificcomponent (heparin when bFGF is used as the mitogen).

Establishment of Multipotent Clonally Derived Sub-Populations of CordBlood Stem Cells.

Isolation of precursor sub-populations based on their response toepigenetic signals generate a homogeneous cell population that behave ina predictable and similar manner when transplanted in vivo or challengedwith a specific treatment in vitro. These clonal cell lines are goodcandidates for clinical-grade development. From the initial startinggrowth factor responsive population of stable human cord blood cells,monoclonal cell populations are established as previously described(Daadi and Weiss, J. Neurosci. 19, 11 4484, 1999). Clusters of cells aredissociated, counted and suspended in media-hormone mix at aconcentration of 1 cell per 15 μl and plated at 15 μl/well in Terasakimicrowells or a 96-well dish. Wells with single cells are immediatelyidentified and marked. Single cells are also randomly picked from thesuspension using a hand-pulled 10 μl micropipette and transferred into aTerasaki microwell containing 10-15 μl of media. Clonal development ismonitored once per day using the inverted microscope with phase-contrastoptics. Cultures are fed by replacing 2 μl with fresh medium every 2days.

Each single cell proliferates and generates a clone of cells. A fractionof these single founder cells have a slow growth rate or do notproliferate or even die after a few days in culture. From our experiencewith neural stem cells and using clonal cultures, some single cells mayundergo cell death because of the lack of neighboring cells that provideextracellular support for cell survival and in specific cases celldivision. If this is a problem, the founder cell are cultured inconditioned medium derived from bulk stem cell cultures, or in thepresence of the membrane extract of cord blood cells.

Characterization and Determination of the Differentiation Efficacy ofEach Clone.

In addition to growing a purified monoclonal human cord blood-derivedstem cell populations, it is necessary to verify that each generation ofthe clone exhibits all stem cell characteristics i.e.: ability to selfrenew, generate a large number of progeny and be able to respond toenvironmental cues and differentiate into different cell types. Theseefficacy criteria are fundamental for the development and the productionof stable multipotent clones. Clonally derived cells (as describedabove) are dissociated either by gentle mechanical trituration or usingtrypsin-EDTA. After the growth phase, part of the next generation cloneis cultured under differentiation conditions. When cells grow as acluster in suspension each clone is removed and plated in controlmedia-hormone mixture without any mitogens on a glass coverslip coatedwith an extracellular matrix (ECM). Different ECMs including laminin,Poly-L-ornithine and poly-D-lysine are tested for their potentialdifferentiation effects. If cells grow as a monolayer, media containingthe mitogen is removed by gentle suction and replaced by control freshmedia (no mitogen). After a culture period of 10 to 15 days,differentiated cells are fixed with paraformaldehyde and stained forvarious neural and, hematopoietic cell markers. Analysis of labeledsubpopulation are carried out using immunocytochemical techniques andFlow Cytometric Analysis. For neural lineages we use: anti-Nestin, andanti-Vimentin to label immature precursor cells; anti-Glial FibrillaryAcidic Protein to label Astrocytes, anti-O4, anti-Myelin Basic Proteinand anti-CNPase to identify oligodendrocytes, Anti-NeuN, Anti-β-tubulinclass III, Anti-Neuron Specific Enolase, Anti-human specificNeurofilament, Anti-MAP2 to identify neurons. Within this last neuronalpopulation we test for different neurotransmitter phenotype expressionlike GABA, Choline Acetyltransferase, Tyrosine Hydroxylase andSerotonin. We also test for hematopoietic cells: Some of thecharacterized multipotent stem cell clones are cryopreserved asdescribed in general methods section (see below) and the rest passagedand maintained in culture.

Cryopreservation

Clonally derived cord blood cells are resuspended in cell freezing mediacomprising 10% dimethyl sulfoxide, 50% Fetal Bovine Serum and 40% ofdefined medium and stored under liquid nitrogen are well known in theart.

The mononuclear layer from whole umbilical cord blood may be preparedfor cryopreservation using the following methodology, which steps may bevaried without significantly changing the cryopreservation outcome.

Processing and Storage of Umbilical Cord Blood

1. Sample Preparation

Anticoagulated cord blood is aliquotted into sterile 50 ml conical tubesand the volume measured accurately. A small sample is removed for whitecell count and sterility testing. A sample of plasma is removed at thistime by centrifugation for cryopreservation. The cord blood is diluted1:2 with sterile phosphate buffered saline (PBS) and mixed carefully toa maximum of 35 ml per tube.

Step 2: Density Gradient Separation

Mononuclear cells are obtained from the cord blood using Ficoll-Hypaquedensity centrifugation. Each tube of diluted cord blood is underlayeredwith 10 ml of sterile Ficoll-Hypaque solution and then centrifuged at1200 g for 30 min. at room temperature. In this procedure, mononuclearcells containing progenitor cells (stem cells) form a layer at theFicoll/plasma interface whereas red cells and granular cells(granulocytes) pass through the gradient to the bottom of the tube. Themononuclear cells are removed carefully by aspiration.

Step 3: Mononuclear Cell Preparation (MNC)

The mononuclear cells are collected in sterile 50 ml tubes and diluted1:2 with tissue culture medium (RPMI) and centrifuged at 1500 g for 15minutes. The cells are further washed in RPMI and resuspended to a fixedvolume (14 ml) and a small sample removed for white cell enumeration andCD34+ cell determination.

Step 4: Preparation of MNC for Cryopreservation

The cell suspension is then centrifuged at 1200 g for 10 mins and thecells resuspended in 2.5 ml of RPMI. A small sample is removed forsterility testing. To this suspension. 2.5 ml of autologous plasmacontaining 10% dimethyl sulfoxide (DMSO) as cryoprotectant is addedslowly and the resulting suspension transferred to a labeled (bar coded)sterile, 5 ml cryovial.

Step 5: Controlled Rate Freezing in Liquid Nitrogen

The samples are then cryopreserved using a controlled rate of freezingfrom 4 deg C. to −90 deg C. using the following protocol:

-   -   +4 degree C. to −3 degree C. at one degree C. per minute    -   −3 degree C. to −20 degree C. at 10 degree C. per minute    -   −20 degree C. to −40 degree C. at one degree C. per minute    -   −40 degree C. to −90 degree C. at 10 degree C. per minute.

The cryovials are then stored in vapor phase of liquid nitrogen at −196degrees C.

Differentiating Culture Conditions

Three T75 flasks of 10-15 days old suspension cultures are spun down for5 min at 400 rpm. The cells are removed and placed into a 12 mlcentrifuge tube and spun down for 5 min at 600 rpm. The growth medium isremoved, and cells resuspended in fresh control media (no EGF or othermitogen) plus hormone mix. This step is repeated one more time to ensurethe complete removal of the mitogen from the media. Dissociated cellsare plated in media hormone mix at a density of 1×10⁶ cells/ml onpoly-L-ornithine-coated (15 μg/ml; Sigma) glass coverslips in 24-wellNunclon culture dishes with 0.5 ml/well. After 7 to 14 days in culture,cells will change morphology into a neuron-like or glial-like phenotype.Following staining with specific antibodies which recognize markers ofneural precursors, of neurons and of glia, the differentiation efficacyof the hormone mix is quantified. The density of positively stained cellbodies are determined in at least 20 randomly selected fields from eachculture dish or well using the 40× objective. For quantitation of totalNeuN-ir, GFAP and nestin-ir cells produced, a total of 3 experiments areperformed resulting in total of 6 culture dishes or wells analyzed foreach condition. Neural differentiation efficacy of a growth factor (orhormone mixture) are calculated as the percentage of NeuN-immunoreactivecells (relative to total number of cells in a dish identified with DAPInuclear stain).

Indirect Immunocytochemistry

Rabbit polyclonal antisera and mouse monoclonal antibodies directedagainst specific antigens are used as primary antibodies for indirectimmunofluorescence. Coverslips fixed with 4% paraformaldehyde for 20 minfollowed by three washes (10 min each) in phosphate buffer saline (PBS).After the PBS rinse, coverslips are processed for single or duallabeling and incubated with primary antibodies generated from differentspecies. The primary antibodies are made in PBS/10% normal goatserum+0.3% triton X-100. After 2 hours incubation at 37° C., thecoverslip are rinsed in PBS. Fluorescent conjugated secondary antibodies(1:100, 1:200, Jackson ImmunoResearch) are applied in PBS for 30 min atroom temperature. Coverslips are then washed three times (10 min each)in PBS, rinsed with water, placed on glass slides, and coverslippedusing Fluorsave (Calbiochem). Fluorescence is detected and photographedusing Zeiss Laser Scanning Confocal microscope (model LSM 510). Theprimary antibodies that are used are against: Nestin (1:1000;PharMingen), Vimentin (1:200, Boehringer), Glial Fibrillary AcidicProtein (1:500, Sigma), O4 (1:100, Chemicon), Myelin Basic Protein(1:200, Boehringer), and CNPase (1:500, Stemberger Monoclonals), NeuN(1:100, Chemicorp), β-tubulin class III (1:1000, Sigma), Neuron SpecificEnolase (1:100, Chemicon), human specific Neurofilament (1:150,Boehringer), MAP2 (1:200, Chemicon), GABA (1:5000, Sigma), CholineAcetyltransferase (1:200, Chemicon), Tyrosine Hydroxylase (1:4000,Incstar), Serotinin (1:200, Chemicon), type IV collagen (1:50, Dako),Laminin (1:300, Sigma), CD10 (1:100, PharMingen), muscle actin (1:1000,Sigma), HLA-DR (1:200, PharMingen), CD45 (1:100, PharMingen), Mac-1(1:100, Chemicon); alkaline phosphatase staining kit (Sigma # 85L-2).

Flow Cytometric Analysis

To assess the actions of specific treatment on differentiation or toestablish a relative profile within a culture condition of differentcell populations labeled with the markers mentioned above, the harvestedcells are subject to Flow Cytometric Analysis. Cells are rinsed withfluorescence activated cell sorting (FACS) buffer (EBSS and 1% HIFBS)and 1×10⁶ cells are added to 100 μl of FACS buffer supplemented with theappropriate primary antibodies and incubated at 4° C. for 30 min. Afterwashing, secondary antibodies are added and incubated at 4° C. for 30min. For biotinylated antibodies, isotope controls are used to setgates; otherwise, gates are set with cells alone. Cell viability ismonitored using propidium iodide exclusion. Flow Cytometric Analysis isperformed with FACScan™ (Becton-Dickinson) with all events gated on theforward and side scatter.

Western Blotting

The culture is washed three times in cold phosphate buffered saline(PBS), scraped into ice-cold PBS, and lysed in ice-cold lysis buffercontaining 20 nM Tris/HCl (pH=8.0), 0.2 mM EDTA, 3% Nonidet P-40, 2 mMorthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl, and10 μg each of aprotinin and leupeptin per ml. After incubation on icefor 10 min, the samples are centrifuged at 14,000×g for 15 min andsupernatants are collected. An aliquot is removed for total proteinestimation (bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample is separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody is blocked with 5% non-fat drymilk overnight at 4° C. Immunoblotting is carried out with theappropriate primary antibody followed by their corresponding peroxidaseconjugated secondary antibodies. The blots are developed by enhancedchemiluminescence method (ECL, Amersham).

Reverse transcription-polymerase chain reaction (RT-PCR) and Northernanalysis Total RNA is extracted using TRIzol (Life Technologies-BRL)according to the recommended protocol.

RT-PCR: aliquot of 1 μg of RNA is reverse-transcribed in the presence of50 mM Tris-HCL, pH 8.3, 75 mM KCL, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTPsand 0.5 μg Oligo-dT (12-18) (Pharmacia) with 200 U Superscript RnaseH-Reverse Transcriptase (Life Technologies-BRL). Aliquots of cDNAequivalent to 40 ng of total RNA are amplified in 25 μl reactionscontaining 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 50 pmol ofeach primer, 400 μM-dNTPs, and 0.5 U AmpliTaq DNA polymerase(Perkin-Elmer). The PCR thermal profile is determined for each pair ofprimer sequence used.

Southern blot. 15 μl aliquots of the amplified PCR products are run on a2% agarose Tris-acetate gel containing 0.5 μg/ml ethidium bromide. Thebands are transferred by capillary action to a Hybond-N+ membrane anddetected using the appropriate radiolabeled probes. The radioactivemembrane is exposed overnight to a BioMaxMR autoradiographic film(Kodak) at −80° C.

Northern hybridization. Aliquots (20 μg) of total RNA are fractioned onagarose formaldehyde gels. The RNA is transferred by capillary actionfrom the gel matrix to Hybond-N+ (Amersham) using 10×SSC, and fixed ontomembrane by baking. These membranes are hybridized with the adequateradio-labeled probes, washed with decreasing concentration of SSC, 0.1%SDS and exposed to a BioMaxMR autoradiographic film for 24 hours (Kodak)at −80° C.

Human Umbilical Cord Blood Contains Multi-Potent Progenitor Cells WhichGive Rise to Neural Lineage

General Methods Following the methods which are generally set forthabove, cord blood was shown to contain cells which can differentiate toneural cells.

Source of Cells

The mononuclear cells for this study were isolated from human umbilicalcord blood samples. The cord blood samples were obtained from the stumpof the umbilical cord on the placental side post-partum. Between 50 and100 ml of blood was obtained per procedure. Cells were spun down,resuspended in cryopreservative medium and frozen in liquid nitrogenuntil needed.

Handling of the Cells and Culture Media. The frozen cells were thawed,spun down, resuspended and plated in 75 mm culture flasks in minimalessential medium (DMEM) supplemented with 2 mM glutamine (100× stockfrom Gibco/BRL), 0.001% B-mercaptoethanol, 1× non-essential amino acids(100× stock from Gibco/BRL) and 10% FBS (stem cell qualified,Gibco/BRL). After 24 to 72 hrs, the medium was replaced with serum free“Neural Proliferation Medium” which consisted of “N2” medium (DMEM/F121:1, Gibco/BRL) supplemented with 0.6% glucose, insulin 25 ug/ml,transferrin 100 ug/ml, progesterone 20 nM, putrescine 60 uM, seleniumchloride 30 nM, glutamine 2 mM, sodium bicarbonate 3 mM, HEPES 5 mM,heparin 2 ug/ml and EGF 20 ng/ml, bFGF 20 ng/ml. Differentiation mediumconsisted of the “Neural Proliferation Medium” without the EGF and bFGF,but instead containing retinoic acid (0.5 μM) plus 100 ng/ml nervegrowth factor (NGF). Detection of proliferating cells was accomplishedby incubating cultures with bromodeoxyuridine (BrdU) (5 μM) for 24-48hrs with subsequent visualization of BrdU immunoreactive cells.

Isolation of RNA. Total RNA was isolated from human cord blood cells (orfractions thereof) using the RNA STAT-60 kit using the protocolrecommended by the manufacturer (Tel-Test “B”, Inc. Friendswood, Tex.77546). Following RNA isolation, its OD density was measured at 260 nm,and stored at −80° C. Integrity was tested on 1% non-denaturing SeakemLE agarose gel (FMC Bioproducts, Rockland, Me.).

DNA Microarray

Total RNA was prepared as above. Total RNA obtained from human cordblood cells, with or without RA+NGF treatment from different batcheswere pooled together for this experiment (15 to 20 g total RNA wasneeded per chip). The human genome U95A array (HG-U95A) from AffymetrixInc. was used in this experiment. The single array represents ˜12,500sequences. The experiment was done at the Functional Genomics Core,Microarray Facility at the H. Lee Moffitt Cancer Center & ResearchInstitute using GeneChip Fluidics station 400, a GeneChip Hybridizationoven, and an HP GeneArray™ scanner. Analysis of the GeneChip microarrayhybridization pattern was performed using GeneChip Analysis Suite 4.0software.

Reverse Transcription (RT). RT was performed using random hexamers asprimers. Final volume was 20 μl with 1 μg of total RNA from eachfraction of cells. The reaction mixture contained 1 mM of eachdeoxynucleoside triphosphate (dNTP), 1 U/μl RNase inhibitor, 5 mM MgCl₂,2.5 U/μl Murine leukemia virus (MuLV) reverse transcriptase, 2.5 μMrandom hexamers in 50 mM KCl and 10 mM Tris-HCl (pH 8.3). It was firstincubated at room temperature for 10 min, and then at 42° C. for 15minutes. The mixture was then be heated at 99° C. for 5 minutes andcooled on ice for 5 min to inactivate the transcriptase.

Polymerase Chain Reaction (PCR). PCR was performed in the same tubes asRT, in 100 μl total volume. Final concentrations were 2 mM MgCl₂, 0.2 mMof each dNTP, and 2.5 U/100 μl Ampli Taq DNA polymerase in the 50 mM KCland 10 mM Tris-HCl buffer (pH 8.3). For generation of various cDNAfragments, a PE 9700 thermocycler (Perkin Elmer, Foster City, Calif.)was programmed as follows: 1 cycle at 95° C. for 105 sec, 35 cycles at95° C. for 15 sec, followed by 60° C. for 30 sec, and finally 1 cycle at72° C. for 7 min. Both RT and PCR was done using Perkin Elmer's GeneAmpRNA PCR kit. To identify the presence of various neuronal markers,primers were constructed based on published human sequences. For Nestin(accession # X65964), forward primer: nt 2524-2542 and reverse primer:nt 2921-2903. For Mushashi-1 (accession # AB012851), forward primer: nt319-339 and reverse primer: nt 618-598. For Necdin (accession #AB007828), forward primer: nt 2374-2393 and reverse primer: nt2767-2747. For Neurofilament subunit NF-L (accession # X05608), forwardprimer: nt 3155-3173 and reverse primer: nt 3521-3501. Primers wereselected using the SEQWEB (version 1.1) software available on the USFcomputer network.

Antibodies

The primary antibodies used included: Mushashi-1 (donated by Prof. H.Okano), Nestin (1:200, Chemicon); NeuN (1:100, Chemicon), class IIIβ-tubulin (1:200, Sigma); GFAP (1:500, Sigma), BrdU (1:400, Chemicon),MAP2 (1:200, Chemicon), pleiotrophin (1:400, R&D Systems).

Western Blot. Cultures were processed using standard methods forperformance of Western blot analysis using the following procedure.Cultures were washed three times in cold phosphate buffered saline(PBS), scraped into ice-cold PBS, and lysed in ice-cold lysis buffercontaining 20 nM Tris/HCl (pH 8.0), 0.2 mM EDTA, 3% Nonidet P-40, 2 mMorthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl, and10 μg each of aprotinin and leupeptin per ml. After incubation on icefor 10 min, the samples were centrifuged at 14,000×g for 15 min andsupernatants were collected. An aliquot was removed for total proteinestimation (Bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample was separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody was blocked with 5% non-fat drymilk overnight at 4 C. The blots were analyzed using the Kodak DS 1DDigital Science Electrophoresis Documentation and Analysis System 120v.0.2.

Immunocytochemistry After 7-14 DIV, the cultures was fixed with 4%paraformaldehyde in 0.1 M phosphate buffer (PB) for 20 minutes. Thecultures were then washed 3 times with phosphate buffered saline priorto beginning immunocytochemistry.

Cell Counts For estimates of cell number in culture, 20 random visualfields (40× objective) in 4 culture dishes for each marker were viewed.The total number of cells visualized under phase contrast microscopy andthe number of positively labeled (immunoreactive) cells was counted ineach visual field. The mean number of labeled cells was then expressedas a percentage of the total number of cells per field.

Results

Cord blood cells, cultured in the presence and absence of retinoic acid(RA) and Nerve Growth Factor (NGF), gave rise to cells bearing neuralprogenitor markers as evidenced by profiles of gene and proteinexpression. A total of 322 genes were either up- or down-regulated by afactor of at least 2, evidenced by measurements using a human microarray“gene chip”. The greatest degree of up-regulation (44 fold increase) wasseen in the mRNA for neurite outgrowth extension protein orpleiotrophin. A significant degree of down regulation was seen in theexpression of tenascin (decreased 8.8 fold), an extracellular matrixprotein that inhibits neurite outgrowth in developing neuronal tissuesand in fibronectin (decreased 5.8 fold), an extracellular matrix proteinthat favors development of blood cell lineages. Other transcriptsassociated with neurogenesis that increased significantly (>2 fold)include glypican-4 (increased 4.9 fold), neuronal pentraxin II(increased 2.3 fold), neuronal growth associated protein 43 (increased2.7 fold), neuronal PAS1 (increased 2.3 fold). Mushashi-1 wasupregulated 1.5 fold. A selection of other genes associated withneurogenesis that were up- or down-regulated is listed in Table I.Concomitant with the increased expression of markers indicative ofneurogenesis, there was a decrease in expression of genes associatedwith hematopoiesis (Table II). The greatest changes occurred in theexpression of HLA class I locus C heavy chain, macrophage receptorMARCO, secreted T cell activation protein Attractin (attractin),leucocyte immunoglobulin-like receptor-8 (LIR-8), thymocyte antigenCD1c, erythropoietin receptor and erythropoietin.

In a parallel set of experiments, total RNA was extracted, and RT-PCRwas performed. The mRNA for nestin and necdin was identified in bothcontrol and RA+NGF treated cultures using primers based on publishedhuman sequences. In each case a product of appropriate length was seenon the gel (FIG. 1A for untreated cells and FIG. 1B for treated cells).Nestin is considered a marker of early neural development, but can alsobe seen in endothelial precursors. Necdin is the gene that codes forneuron specific nuclear protein. The m-RNA for Mushashi-1, the earliestmarker of neural precursors was detected in RA+NGF treated cultures andminimally detected in DMEM-treated controls. The mRNA for neuriteoutgrowth promoting protein (pleiotrophin) was detected in RA+NGFtreated cells, but the signal was much weaker in untreated cultures(FIG. 1C). The mRNA for glypican-4 was detected under both conditions.The mRNA for GFAP, a marker of astrological cells, was also detectedunder both conditions, though the signal was stronger in the RA+NGFtreated cells. No messenger RNA for neurofilament subunit NF-L wasdetected in either treated or untreated cells although it was seen to beup-regulated in the microarray. A negative RT control (without reversetranscriptase) was run with all the reactions to check for genomic DNAcontamination in the RNA preparation while human-actin primers(Clontech) were used as a positive control. We also tested primers forMushashi-1 and Neurofilament subunit NF-L using human brain RNA(Clontech) by RT-PCR; these each generated a single band of appropriatelength (data not shown).

Microscopic examination of immunostained cultures treated with RA+NGFrevealed a heterogeneous mixture of cell types ranging from large flatepitheloid cells to small spindle-shaped cells with fine branchingneuritic processes. A significant proportion (5-10%) of the small cellsin the RA+NGF treated cultures, but not control cultures treated withDMEM, were Mushashi-1 immunoreactive (See FIG. 2A). A similar proportion(5%) of the small cells exhibited β-tubulin III immunoreactivity (FIG. 2B). Approximately 50% of the cells were immunoreactive for BrdU,indicating that the cells were continuing to proliferate (data notshown). Antibodies to nestin (purchased from Chemicon, raised againstrat nestin) failed to recognize the human form of nestin, though theyhad been shown to react with rat nestin in rat bone marrow-derivedneural progenitor cells (Sanchez-Ramos, et al., Neuroscience News 3,32-43, 2000). Approximately 50% of RA+NGF treated cells wereimmunoreactive for GFAP, a marker of astrocytes (FIG. 2E). Western blotsof the cultures confirmed the presence of Mushashi-1 protein,β-tubulin-III protein, pleiotrophin, GFAP and NeuN in both treated anduntreated cells. Densitometric analysis of the blots showed that NGF+RAtreatment increased protein expression (relative to β-actin) ofMushashi-1, β-tubulin-III, pleiotrophin and NeuN (Table III). TABLE IExpression of Genes Associated with Neurogenesis Gene transcript Foldchange following RA + NGF Neurite outgrowth-promoting protein +44extracellular matrix-associated protein that enhances axonal growth inperinatal cerebral neurons [Raulo, 1992 #384] Glypican-4 +4.9 glypican-4is expressed in cells immunoreactive for nestin and the D1.1 antigen,markers of neural precursor cells. Glypican-4 expression not detected inearly postmitotic or fully differentiated neurons [Hagihara, 2000 #375]β-tubulin folding cofactor D ˜+4.6 Pro-galanin +3.9 Found in neurons ofarcuate nucleus of hypothalamus FE65 stat-like protein ˜+3.8 the exon9-inclusive (E9) form is exclusively expressed in neurons[Hu, 1999 #369]Glial acidic fibrillary protein ˜+3.2 Neuron derived orphan receptor˜+2.2 Neuronal pentraxin II (NPTX2) ˜+2.3 member of a new family ofproteins identified through interaction with a presynaptic snake venomtoxin taipoxin; may function during synapse formation and remodeling[Kirkpatrick, 2000 #393] Neuronal growth protein 43 (GAP-43) ˜+2.7Identifies neurons, but also developing muscle cells[Moos, 1993 #395]Neuronal PAS1 (NPAS1) +2.3 transcription factors selectively expressedin the central nervous system [Zhou, 1997 #394] Neuronal DHP-sensitive,voltage-dependent, +2.1 calcium channel alpha-1D subunit Bonemorphogenetic protein 1 (BMP-1) +2 BMP-1/Tolloid is found at the neuralplate/ectodermal transition. Expression is maintained in thepremigratory neural crest, and transiently in the migrating cephalicneural crest cells. [Marti, 2000 #391] Retinal glutamate transporterEAAT5 ˜+2 TrkC ˜+2 Receptor for neurotrophin-3 (NT3) ENO2 gene forneuron specific (gamma) +1.9 enolase Human brain protein recognized bythe sera of ˜+1.8 patients with paraneoplastic sensory neuronopathy Bonemorphogenetic protein 2A ˜+1.8 Neuronal PAS2 (NPAS2) +1.7 transcriptionfactors selectively expressed in the central nervous system [Zhou, 1997#394] Survival motor neuron pseudogene +1.7 Glial Growth Factor 2 +1.6Neural cell adhesion molecule (N-CAM) Exon ˜+1.6 SEC Follistatin-relatedprotein (FRP) +1.6 Microtubule-associated protein 2 (MAP2) ˜1.6Vesicular acetylcholine transporter +1.6 Neurofilament subunit M (NF-M)+1.5 Neurofilament subunit NF-L +1.5 Musashi1 ˜1.5 Bone morphogeneticprotein 11 (BMP11) +1.5 BMP-11 is expressed in the developing nervoussystem; at higher doses induces nervous tissue [Gamer, 1999 #392]Tenascin-C −8.8 Tenascin-C is an extracellular matrix protein thatinhibits neurite extension, and promotes cell proliferation andmigration [Thomas, 1996 #397; Anstrom, 1996 #398]

TABLE II Downregulation of Genes Associated with Development of BloodLines Fold decrease HLA class I locus C heavy chain −6.4 Macrophagereceptor MARCO −4.9 secreted T cell activation protein Attractin −3.6(attractin) alpha-1 collagen type II −3.0 Leucocyte immunoglobulin-likereceptor-8 −2.8 (LIR-8) Thymocyte antigen CD1c −2.5 Erythropoietinreceptor ˜−2.6 Erythropoietin ˜−2.4 Monocyte chemotactic protein-2 −2.2LAG-3 mRNA for CD4-related protein involved ˜−2.3 in lymphocyteactivation Interleukin-7 receptor (IL-7) −2.2 Complement receptor type 1−2.1 T cell receptor −2 p50-NF-kappa B homolog −2 Lymphocyte-specificprotein tyrosine kinase ˜−2 (LCK) LAG-3 mRNA for CD4-related proteininvolved ˜−2.3 in lymphocyte activation Erythrocyte membrane proteinRh30A (Rhesus ˜−2.1 antigen) Erythrocyte membrane protein band 4.2 ˜−2.9(EPB42) Leukocyte IgG receptor (Fc-gamma-R) −1.8 Erythroblast macrophageprotein EMP −1.5

TABLE III Densitometric Measurement of Expressed Proteins Separated byWestern Blot Ratio to Ratio to MW β-Actin β-Actin Protein marker DMEMNGF % change Musashi-1 36 kD 0.805 0.93 +15.5% β-III tubulin 75-80 kD0.328 0.50 +52.4% Pleiotrophin 18 kD 0.179 0.315 +75.9% GFAP 46 kD 0.5380.515  −4.3% NeuN 51 kD 0.6 0.715 +19.1% β-Actin 42 kDDiscussion of Results

These findings demonstrate that human umbilical cord blood containscells that can be induced to express markers of neural development,including Mushashi-1, glypican-4 and β-tubulin III. Recent work hasdemonstrated Mushashi-1 immunoreactivity in the developing and/or adultCNS tissues of frogs, birds, rodents, and humans (Kaneko, et al.,Developmental Neuroscience 22, 139-53 (2000). The anti-Mushashi-1monoclonal antibody has been shown to react with undifferentiated,proliferative cells of the sub-ventricular zone in the CNS of allvertebrates tested. Glypican-4, upregulated five-fold in the cord bloodcultures treated with RA+NGF, has been reported to be expressed in cellsimmunoreactive for nestin and the D1.1 antigen, other known markers ofneural precursor cells, but it has not been detected in earlypostmitotic or fully differentiated neurons (Hagihara, et al,Developmental Dynamics 219, 353-67 (2000). β-tubulin III is one of themost specialized tubulins specific for neurons (Fanarraga, et al.,European Journal of Neuroscience 11, 517-27 (1999). Both theupregulation and the post-translational processing of class-IIIbeta-tubulin are believed to be essential throughout neuronaldifferentiation (Laferriere, et al., Cell Motility & the Cytoskeleton35, 188-99 (1996) and Laferriere, et al., Biochemistry & Cell Biology75, 103-17 (1997).

The cord blood cultures treated with RA+NGF also increased expression ofmany genes specific for neurons including pentraxin II, GAP43, FE65stat-like protein, neuronal PAS1 and PAS2. Neuronal pentraxin II is amember of a new family of proteins identified through interaction with apresynaptic snake venom toxin taipoxin. Neuronal-pentraxin-II mayfunction during synapse formation and remodeling (Kirkpatrick, et al,Journal of Biological Chemistry 275, 17786-92 (2000). Neuronal growthassociated protein 43 (GAP43) is considered a specific neuronal markerbut may also be expressed in developing myocytes (Moos, T. &Christensen, L. R. GAP43 identifies developing muscle cells in humanembryos. Neuroreport 4, 1299-302 (1993). FE65 stat-like protein (theexon 9-inclusive form) is specifically expressed in neurons (Hu, et al.,Journal of Neuroscience Research 58, 632-40 (1999). Neuronal PAS1 andPAS2 are transcription factors selectively expressed in the centralnervous system (Zhou, et al., Proceedings of the National Academy ofSciences of the United States of America 94, 713-8 (1997). Other genesindicative of neurogenesis that were expressed following treatmentincluded the neurofilament subunits-NF-L and NF-M, microtubuleassociated protein 2 (MAP2), the vesicular acetyl choline transporter,and neuronal DHP-sensitive, voltage-dependent, calcium channel alpha-1Dsubunit. Cord blood cells expressed mRNA for neuronal specific enolase,but this protein is also expressed by many cells in bone marrow,especially megakaryocytes. The greatest change observed in cord bloodcultures treated with RA+NGF was a 44 fold increase in expression ofmRNA for an extracellular matrix-associated protein that enhances axonalgrowth in perinatal cerebral neurons (Raulo, et al., Journal ofBiological Chemistry 267, 11408-16 (1992). At the same time there was asignificant decrease in expression of mRNA for tenascin, anextracellular matrix protein which inhibits neurite outgrowth (Kukekov,et al., Experimental Neurology 156, 333-44 (1999). There was alsoevidence for glial cell development. Increased expression of the glialcell marker GFAP was measured in the microchip data, and confirmed byimmunocytochemistry. Concomitant with the increased expression ofmarkers indicative of neurogenesis, there was a decrease in expressionof genes associated with development of blood cell lines.

The present findings provide evidence that cord blood contains amulti-potent cell capable of differentiating into a neural lineage. Theease with which the umbilical cord blood can be obtained, stored, andexpanded in culture could make this a preferable source of cells fortransplantation for neurodegenerative diseases, gene delivery to thecentral nervous system, and repair of brain and spinal cord injuries.

Example Identification and Isolation of Mononuclear Cells ExpressingNeuronal, Astrocytic or Oligodendrocytic Markers and Use of MononuclearCells to Effect Transplantation in Stroke

This series of experiments is directed to using both cell culturetechniques and an animal model of cerebral ischemia to establish humancord blood as a viable source of NSCs for the treatment of CNS diseaseor injury. These studies determine the existence of mononuclear cells incord blood that express neuronal, astrocytic or oligodendrocytic markersand identify those mononuclear cells that give rise to neural celllineages. The cord blood stem cells are shown to provide a stable,readily available source of NSCs which become functional neurons and arecapable of producing behavioral recovery at a comparable level to thatobserved with transplantation of fetal neurons.

General Methods.

Culture Media. The frozen cells are thawed, spun down and resuspendedand plated in 75 mm culture flasks in minimal essential medium (DMEM)supplemented with 2 mM glutamine (100× stock from Gibco/BRL), 0.001%B-mercaptoethanol, 1× non-essential amino acids (100× stock fromGibco/BRL) and 10% FBS (stem cell qualified, Gibco/BRL). After 24 to 72hrs, the medium is replaced with serum free “Neural ProliferationMedium” which consists of N2 medium (DMEM/F12 1:1, Gibco/BRL)supplemented with 0.6% glucose, insulin 25 ug/ml, transferrin 100 ug/ml,progesterone 20 nM, putrescine 60 uM, selenium chloride 30 nM, glutamine2 mM, sodium bicarbonate 3 mM, HEPES 5 mM, heparin 2 ug/ml and EGF 20ng/ml, bFGF 20 ng/ml. Differentiation medium consists of the “NeuralProliferation Medium” without the EGF and bFGF, but instead containingretinoic acid (0.5 μM) plus a specific growth factor (NGF, BDNF, orGDNF)

Transfection of Cord Blood Cells with Fluorescent Green Protein Drivenby the Mushashi-1 Promoter.

An ΔE1 adenovirus bearing hGFP under the control of the Mushashi-1promoter (AdP/Mushashi) (generously donated by H. Okano of Japan) areused to transfect umbilical cord blood cells. This adenoviral DNA vectoris a plasmid DNA that contains a portion of the viral genome in whichthe E1 A region is deleted and the hGFP under control of the Mushashi-1promoter has been inserted in the place of the E1A region of the genome.Cells to be transfected are plated in 0.5 ml of serum-free “NeuralProliferation Medium”. To each culture dish of cells to be transfected0.8 μg of the DNA is diluted and mixed into 50 μl of Opti-Mem® I ReducedSerum Medium Without Serum (Life Technologies, Inc). Eight μl of PlusReagent Mix is added and incubated at room temperature for 15 min.Lipofectin Reagent (Life Tech, Inc) is diluted and mixed in a secondtube (0.5 μl into 50 μl of Opti-Mem I Reduced Serum Medium WithoutSerum). After 30 min incubation at room temperature, the precomplexedDNA is mixed with diluted Lipofectin Reagent and incubated for 15 min atroom temperature. Then the DNAPlus-Lipofectin Reagent complexes (100 μl)are added to each well and mixed gently by rocking the plate back andforth. The cultures are incubated at 37° C. in 5% CO2 for 4-5 h. After24 to 48 hrs, selected cultures are harvested to assess efficiency oftransfection.

Isolation of RNA. Total RNA is isolated from human cord blood cells (orfractions thereof) using the RNA STAT-60 kit using the protocolrecommended by the manufacturer (Tel-Test “B”, Inc. Friendswood, Tex.77546). Following RNA isolation, its OD density is measured at 260 nm,and stored at −80° C. Integrity is tested on 1% non-denaturing Seakem LEagarose gel (FMC Bioproducts, Rockland, Me.).

Reverse Transcription (RT). RT is performed using random hexamers asprimers. Final volume is 20 μl with 1 μg of total RNA from each fractionof cells. The reaction mixture contains 1 mM of each deoxynucleosidetriphosphate (dNTP), 1 U/μl RNase inhibitor, 5 mM MgCl₂, 2.5 U/μl Murineleukemia virus (MuLV) reverse transcriptase, 2.5 μM random hexamers in50 mM KCl and 10 mM Tris-HCl (pH 8.3). It will first be incubated atroom temperature for 10 min, and then at 42° C. for 15 minutes. Themixture will then be heated at 99° C. for 5 minutes and cooled on icefor 5 min to inactivate the transcriptase.

Polymerase Chain Reaction (PCR). PCR is performed in the same tubes asRT, in 100 μl total volume. Final concentrations are 2 mM MgCl₂, 0.2 mMof each dNTP, and 2.5 U/100 μl Ampli Taq DNA polymerase in the 50 mM KCland 10 mM Tris-HCl buffer (pH 8.3). For generation of various cDNAfragments, a PE 9700 thermocycler (Perkin Elmer, Foster City, Calif.) isprogrammed as follows: 1 cycle at 95° C. for 105 sec. 35 cycles at 95°C. for 15 sec, followed by 60° C. for 30 sec, and finally 1 cycle at 72°C. for 7 min. Both RT and PCR are done using Perkin Elmer's GeneAmp RNAPCR kit. To identify the presence of various neuronal markers, primersare constructed based on published human sequences. For Nestin(accession # X65964), forward primer: nt 2524-2542 and reverse primer:nt 2921-2903. For Mushashi-1 (accession # AB012851), forward primer: nt319-339 and reverse primer: nt 618-598. For Necdin (accession #AB007828), forward primer: nt 2374-2393 and reverse primer: nt2767-2747. For Neurofilament subunit NF-L (accession # X05608), forwardprimer: nt 3155-3173 and reverse primer: nt 3521-3501. Primers wereselected using the SEQWEB (version 1.1) software available on the USFcomputer network.

Western Blot. Cultures are washed three times in cold phosphate bufferedsaline (PBS), scraped into ice-cold PBS, and lysed in ice-cold lysisbuffer containing 20 nM Tris/HCl (pH=8.0), 0.2 mM EDTA, 3% Nonidet P-40,2 mM orthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 100 mM NaCl,and 10 μg each of aprotinin and leupeptin per ml. After incubation onice for 10 min, the samples are centrifuged at 14,000×g for 15 min andsupernatants are collected. An aliquot is removed for total proteinestimation (Bio-Rad assay). An aliquot corresponding to 10 μg of totalprotein of each sample is separated by SDS/PAGE (10%) under reducingconditions and transferred electrophoretically to nitrocellulosefilters. Nonspecific binding of antibody is blocked with 5% non-fat drymilk overnight at 4 C.

MCAO Induction. Sprague Dawley rats are anesthetized with Isofluoraneand an incision made from the caudal end of the sternomastoid andsternothyroid muscles extending toward the ears. Using blunt dissectiontechniques, the right common carotid artery is exposed and carefullydissected free of the vagus nerve. The external carotid will then betied off and an embolus (a 40 cm length of 4.0 monofilament) is insertedthrough the external carotid approximately 25 mm into the internalcarotid. At this point, the embolus is blocking the origin of the rightmiddle cerebral artery. The embolus is left in place for 1 hr. Afterremoval, the external carotid is cauterized and the incision closed. Theanimals are allowed to recover for 24 hr prior to transplantation.

Transplantation. The freshly isolated MNCs are resuspended in HBSS+15 mMHEPES at a cell concentration of 100,000 cells/μl. The coordinates forthe injection site are 1.2 mm anterior and +2.7 mm lateral to the bregmaand −5.2 and −4.7 mm ventral to the dura with the toothbar set at zero.Five microliters of the cell suspension are deposited at 2 sites in thestriatum adjacent to the infarct site along a single needle tract. Eachinjection of 2.5 μl is delivered at the rate of 1 μl/min. The needle isleft in place for an additional 5 min after the injection and thenwithdrawn slowly. The incision is closed with wound clips. For thetransplantation of the expanded and/or differentiated MNCs, the cellsare lifted from the culture flasks with gentle mechanical trituration orlifted with trypsin (0.25%) and 1 mM EDTA at 37 C for 3-4 min and washedthree times with HBSS+15 mM HEPES. Cell concentration is adjusted to100,000 cells/μl.

Behavioral testing methods. Twenty-four hours after stroke, the animalsundergo a standardized neurological screening exam measuring 5 motor andpostural activities to verify the extent of the MCAO damage. Thisbattery is repeated at one month post stroke. In addition, the animalsare tested at both time points in the Passive Avoidance test of learningand memory. In the acquisition phase of the test, the animal is placedon a platform in the corner of a Plexiglas cage. When it steps off theplatform, the rat will receive a scrambled foot shock (approximately 2mA) for as long as it remains off the platform. Learning is measured bythe amount of time required for the rat to remain on the platformcontinuously for 3 minutes, and the number of times it leaves theplatform. Twenty-four hours later, retention is measured by placing therat on the platform, and recording the latency to step-down measured toa maximum of 3 min and the number of step-downs. Animals are also testedin the Rotorod Test of motor coordination. The animal is placed on arevolving rod (16 rpm) and the latency to the first fall as well as thenumber of falls in a 3 minute test is recorded. The test is repeatedtwice for a total of 3 tests per testing session with a minimum 30 min.rest between tests. The third behavioral observation includesSpontaneous Activity Monitoring. The animals are placed in a squareacrylic box overnight with an infrared grid to measure movement anddirection. The Elevated Body Swing Test, a measure of motor asymmetry isalso performed. The animal is held by the base of the tail and lifted 2″above the base of the cage. The direction that the head and body islifted is recorded. The test is repeated 20 times. The final test isSkilled Forepaw Use. This is also a measure of motor asymmetry. Theanimal is placed in an acrylic chamber with two descending staircases.Each step is baited with 5 food pellets. The chamber is designed suchthat each staircase can only be reached by one paw. The number ofpellets retrieved measures the function of each limb. There is a 5 daytraining period, during which the animals are partially food deprived.All behavioral data are reported as mean±sem.

Tissue Preparation in Culture Preparations. After 7-14 DIV, the culturesare fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (PB) for 20minutes. The cultures are then washed 3 times with phosphate bufferedsaline prior to beginning immunocytochemistry.

Tissue Preparation of Brain Sections. The rats are sacrificed under deepchloral hydrate (10%) anesthesia and transcardial perfusion of the brainwith 50 ml of 0.1 M phosphate buffer (PB) and then 250 ml 4%paraformaldehyde in 0.1 M PB performed. The brain is removed, post-fixedfor 24 hr and cryopreserved in 20% sucrose prior to cutting 30 μm thickfrozen sections through the forebrain.

Immunohistochemistry. Single and double immunofluorescencehistochemistry are performed. Briefly, the floating sections will firstbe quenched by incubation in a 10% methanol, 3% hydrogen peroxidesolution in phosphate buffered saline (PBS) followed by pre-incubationin 10% normal serum (horse or goat; Vector) in 0.3% Triton-X100 (Sigma)in PBS. The sections are transferred to primary antibody in 2% normalserum, 0.3% Triton X-100/PBS and incubated overnight at 4° C. Theprimary antibodies that are used include: Mushashi-1 (donated by Prof.H. Okano), Nestin (1:200, Chemi-Con); vimentin (1:500 Chemi-Con) asmarkers of early neural precursors; NeuN (1:100, Chemi-Con) and Hu (1:20Molecular Probes) class III β-tubulin (1:200, Sigma) to identify humanneurons at specific stages of development; human specific GFAP (1:200,Stemberger Monoclonals) to identify astrocytes; and O4 or 2′3′ cyclicnucleotide 3′ phosphodiesterase (CNPase, 1:200, Sigma) to identifyoligodendrocytes derived from the transplanted human MNCs. The sectionsare then washed in PBS before being placed in secondary antibodyconjugated to either fluorescein or rhodamine for 2 hours. The sectionsare rinsed in PBS, mounted and coverslipped with Vectashield.Confirmation that a cell is doubly-stained with be obtained byz-stacking analysis of images collected with a Zeiss ConfocalMicroscopes (LSM 510).

Cell Counts. For assessment of cell number in culture, 20 random visualfields (40× objective) in 4 culture dishes for each marker in 3replicates are viewed. The total number of cells and the number ofpositively labeled cells are counted. For each experimental condition,the number of positive cells and the total number of cell nuclei stainedwith 4′,6-dimidinee-2′-phenylindole dihydrochloride (DAPI) aredetermined. The total counts are then expressed as a percentage of thetotal DAPI-stained nuclei. For quantification of immunofluorescence inbrain sections, an unbiased counting methodology are used. Neurons aredirectly counted in a small number of sections at predetermined uniformintervals for the entire set of sections containing specific CNS nuclei.Within each section to be counted the field of view is focused at thetop of the section using a 40× objective. The focus is then shiftedthrough the section and the number of positive profiles not present atthe top of the section is counted.

Analysis. The number of animals to be used in these studies was based ona power analysis of data obtained in previous experiments in thislaboratory. The analysis showed that a minimum of 10 animals per groupis needed to find a difference in the variables of interest at asignificance level of p<0.05. All quantifiable results are expressed asmean±sem and are analyzed using Analysis of Variance (ANOVA). Allpost-hoc tests are conducted using a Scheffé test.

Identification and Isolation of Stem/progenitor Cells Present inUmbilical Cord Blood

The ideal way to identify and isolate neural progenitor cells among theheterogeneous population of mononuclear cord blood cells is to utilize acell surface marker to which a fluorescent or magnetic bead antibody tagis attached to facilitate sorting and separation. Unfortunately, bothNestin and Mushashi-1 are located in the cytoplasm. Surface-basedimmunoselection strategies do not yet permit the prospectiveidentification or specific extraction of neural stem/progenitor cells. Anovel strategy has been used to identify and monitor internal molecularmarkers of neural progenitor cells and to separate the neuralprogenitors from other cells using fluorescence activated cell sorting(FACS) (N. S. Roy et al., Journal of Neuroscience Research 59, 321-31(2000)). This method relies on coupling the promoters required forneuroepithelial-specific gene expression with a reporter gene (eitherlacZ or Green Fluorescent Protein-GFP). More specifically,cis-regulatory elements (the “promoter”) required for the expression ofMushashi-1 or α-tubulin-1 were placed upstream to the reporter gene GFP(Wang et al., Nature Biotechnology 16, 196-201 (1998) and N. S. Roy etal., Journal of Neuroscience Research 59, 321-31 (2000)) Using thisapproach, neural progenitors and young neurons have been identified andselectively harvested from a variety of heterogeneous samples, includingboth adult and fetal mammalian forebrains at different developmentalstages (Wang, et al., supra, and Keyoung et al., Society forNeuroscience Abstract, 159 (2000)).

Experimental Design. We identify and separate neural progenitor cells byFACS of cord blood cells transfected with the gene for GFP, driven bythe neuronal promoter α-tubulin-1 (Tα1) or by the Mushashi-1 promoter.Mononuclear cells are obtained from the placental stump of the umbilicalcord after delivery and processed by Ficoll centrifugation (See GeneralMethods). This results in nearly 100% recovery of mononuclear cells.These cells are cryopreserved in aliquots of 2 million cells until theyare to be used. After thawing, and plating in culture flasks insupplemented minimal essential medium (DMEM) plus FBS 10% for 48 hrs,the medium is changed to “Neural Progenitor Proliferation Medium” for 2days (See Methods for definition of the media). Then, the mononuclearcells are transfected in a suspension culture with a plasmid or viralvector containing the gene for GFP under the control of P/Tα1 orMushashi-1 (See General Methods for details on Transfection techniqueand description of the vectors). After a 6-hour transfection, the cellsare spun down, resuspended in “Neural Progenitor Proliferation Medium”and plated in small culture flasks (See General Methods). GFP shouldtypically be expressed by appropriate target cells within 2 days oftransfection. Flow cytometry and sorting of GFP+ cells are performedafter 2-7 days in culture. Cells are washed, dissociated and analyzed bylight forward and right-angle (side) scatter, and for GFP fluorescencethrough a 510±20 nm band pass filter as they traverse the beam of theLaser (488 nm, 100 mW). Sorting is done using a purification-modealgorithm. Cells detected as being more fluorescent than background aresorted at 1,000-3,000 cells/s. Sorted GFP+ cells are plated in 24 wellculture plates in “Neural Proliferation Medium” (See General Methods fordetails) and BrdU. At 2 and 7 days post-FACS, the sorted cultures arefixed and immunostained for BrdU together with either Mushashi-1,β-tubulin-III, Nestin, NeuN, MAP2, glial fibrillary acidic protein(GFAP) or O4 (to detect oligodendrocytes).

Results and Alternative Method for Enriching Stem-like Cells. Cellstransfected with the plasmid DNA encoding P/Tα1:GFP or the viral vectorencoding P/Mushashi:GFP identify neural progenitors and immature neuronsas evidenced by immunoreactivity for Mushashi-1, β-tubulin-III, andNestin. In most instances, at least 50% of the Mushashi-1(+) cells areco-labeled with BrdU antibody indicating that the cells areproliferating. Based on the work of Wang et al., supra, manyMushashi1-driven GFP+ cells should be labeled for up to 7 to 10 days.These neural progenitors and their daughters should be selected andsubstantially enriched by FACS. Further, they also showed that 0.36% ofadult ventricular zone dissociates expressed Tα1-driven GFP. Assumingthat only 0.01% of cord blood cells express Mushashi1-driven GFP, andthe transfection efficiency using the plasmid DNA is 12.5%, we estimatethat for every 5×10⁶ cord blood cells processed, we shall obtain625-1000 neural precursor cells. However, using an adenoviral vectorresults in a much greater transfection efficiency. For that reason wealso use a ΔE1 adenovirus bearing hGFP under the control of the Mushashipromoter (AdP/Mushashi:hGFP) provided generously by H. Okano of Japan.

In an alternative method, we first identify the least committedstem-like cells from the mononuclear cells in the cord blood and isolatethis population from both CD34+ and CD34− cells prior to inducingproliferation and promoter-based isolation of neural progenitors. Thebasic premise of this strategy is that stem cells are quiescent andexpress very few cell surface markers, except during a proliferationphase. Primitive stem cells fail to stain with Hoescht 33342 and PyroninY and can be separated on this basis using FACS. Further, separation ofcells that express P-gp, the transmembrane protein product of themultiple drug resistance gene (MDR), which is likely to be expressed incells that exhibit characteristics of stem cells, could also beperformed. These staining characteristics could be used to separate stemcells from cord blood mononuclear cells using FACS. If FACS demonstratesthat P-gp+ cells are also cells that fail to stain with the fluorescentdyes (Hoescht 33342 and Pyronin Y), then magnetic bead cell sorting areused to physically separate the P-gp immunoreactive cells from cordblood. In order to increase the yield of neural progenitor cells, it ispreferable to start with the smaller population of the least committedcells found in cord blood.

Assessing the Self-Renewal Capacity of the Neural Progenitor Population

In the example above, we identify a subpopulation of cells enriched withneural progenitor cells or uncommitted stem cells. It will, therefore,be critical to expand the cell populations in order to obtain sufficientcells for study and eventually for transplantation. In this study wewill determine whether there are differences in the ability of theisolated populations of cells to proliferate and the best agents forinducing proliferation in the neural progenitors.

Experimental Design. Mononuclear cells are obtained and thesubpopulations isolated as described above. We will focus primarily onthe GFP+ cells containing neural progenitors. These cells are plated inCorning T75 flasks with “Neural Progenitor Proliferation Medium”. Thisserum-free, defined medium contains epidermal growth factor (EGF) andbasic fibroblast growth factor (bFGF) and is used to induceproliferation of neural stem cells derived from fetal or adult brains.Once the cultures reach confluence (about 1 week), the cells are liftedby incubation with 0.25% trypsin, and 1 mM EDTA for 3-4 minutes. Analiquot of cells is replated with BrdU to assess the proportion of cellsthat are actively proliferating. The cells are replated after 1:3dilution with Neural Progenitor Proliferation medium. Cell yield andviability is also determined with the trypan blue dye exclusion assayafter each passage, for at least five passages.

Results. Neural stem cells proliferate with exposure to EGF or bFGF andthe combination of these growth factors optimally allow for thecontinuous, rapid expansion and passaging of human neural stem cells.Alternatively, there is an extensive list of trophic factors andcytokines that may be more or less effective in inducing proliferation.These include other members of the EGF family such as transforminggrowth factor (TGF)-, amphiregulin, betacellulin and heregulin; FGF2 andthe related FGF1 and FGF4, platelet-derived growth factor family (PDGF),interleukins, and members of the TGF β superfamily. There may be somedegree of differentiation that occurs despite culturing in presence ofknown mitogens. The proportion of cells that continue to proliferate(determined by the BrdU assay before each passage) will guide us in theselection of the optimal mitogens for the neural progenitors.

Assessing the Capacity of Cord Blood Derived Neural Progenitors toDifferentiate into Neurons, Astrocytes or Oligodendrocytes In Vitro.

The mononuclear cells which proliferate subpopulations of mononuclearcells as established above can differentiate into neurons, astrocytesand oligodendrocytes. We have shown that a small population ofnon-hematopoietic stem cells in the bone marrow stromal cell fractionwill differentiate into neurons and astrocytes. Moreover, preliminaryresults show that cord blood treated with RA+NGF for less than one weekexpress a marker seen in early neuronal development, β-tubulin-III. Inthis study, we demonstrate that treatment with “Differentiation Media”will drive our neural progenitors into neuronal and glial phenotypes.

Experimental Design. Mononuclear cells from the first or second passageof the subpopulations with the greatest proliferative capacity asdetermined above (GFP+ cells) are replated in 35 mm culture dishes inthe presence of a “neuronal differentiation medium” (See Methods fordefinition) and a series of specific neurotrophic factors. The first tobe tried are brain derived neurotrophic factor (BDNF, 10 ng/ml) sincethis media has been used previously with bone marrow stromal cells todifferentiate the cells along neural lineages. After 7-14 days in vitro(DIV), cultures are processed for Western blotting, RT-PCR andimmunocytochemistry to identify cells that express neural markers. Themarkers to be examined include nestin, vimentin, glial fibrillary acidicprotein (GFAP) to label astrocytes, O4, myelin basic protein and CNPaseto identify oligodendrocytes and NeuN, β-tubulin class III, Hu,Neuron-Specific Nuclear antigen (NeuN), human specific neurofilament andmicrotubule associated protein (MAP-2) to identify neurons.Quantification is described in the General Methods.

Results. Neural markers are observed in the subpopulations chosen forassay based on preliminary results with cord blood and results obtainedwith differentiation of bone marrow stromal cells. Further, thepopulation most likely to give rise to these neural cells is theGFP-expressing cells (driven by the Mushashi-1 promoter). Alternatively,it may be necessary to use the stem-like cells obtained by selecting theleast committed of cells from the cord blood, nonetheless we are stillable to obtain significant numbers of differentiated neurons and gliafollowing treatment with differentiation media.

Example Expanded Population of Mononuclear Cells and Expression ofNeural Markers after Transplantation into Middle Cerebral ArteryOcclusion (MCAO) Model of Stroke

In addition to demonstrating the existence of the neural phenotype invitro, it is important to show that the isolated and differentiatedcells could express or maintain their neural phenotype aftertransplantation. The culture environment is easily controlled andmanipulated. The environment into which the cells are transplanted invivo is much less predictable; and there are many influences on thecells once they are placed in situ, some of which may alter theproliferative capacity or phenotypic lineage of the cell. Therefore, wedemonstrate that these stem-like cells maintain the ability to becomeneurons, astrocytes or oligodendrocytes in the brain.

Experimental Design. Sprague Dawley rats (n=10/group) are assigned toone of the following groups: 1) Middle cerebral artery occlusion (MCAO);2) MCAO with a striatal transplant of freshly isolated mononuclearcells; 3) and 4) MCAO+expanded GFP+ cells from the two subpopulationswith the highest proliferative capacity as determined above; 5) and 6)MCAO+expanded/minimally differentiated GFP+ cells as determined above.The untreated cord blood cells in group 2 are labeled with thefluorescent dye PKH26 for later identification in the brain and thentransplanted into the striatum in the penumbral region of the infarct.(The isolated neural progenitors will already be labeled by the GFP).The animals are evaluated on a series of behavioral measures and aneurological exam at 24 hr and one month. This includes two paradigms wehave used to demonstrate behavioral deficits after stroke and recoveryfollowing transplantation, the passive avoidance test of cognitivefunction and the rotorod test of motor coordination. The animals willthen be perfused with 4% paraformaldehyde and the brains harvested forhistological and immunohistochemical analysis of graft survival andneural differentiation. Sections are examined for the presence ofPKH26-positive (or GFP+) cells, and cells that express human NuclearMatrix Antigen (NuMA), allowing a second method of identifying humancord blood-derived cells in the rat brain). Other sections aredouble-labeled for PKH26 or NuMA and Hu, class III β-tubulin, NeuN,GFAP, O4. The proportion (%) of cord blood derived cells (NuMA-ir andPKH26+) that express neuronal markers (NeuN, Hu, or class IIIβ-tubulin), glial markers (GFAP) and oligodendrocyte markers (O4) aredetermined.

Results. Based on our preliminary results we see both behavioralimprovements and surviving stem-cell progeny in the MCAO-injured brain.Behavioral improvements are observed in all transplant groups, althoughit is better in the expanded/minimally differentiated cells where theexpanded cell population have already been committed to a neural lineageand therefore may be expected to express more neurons. With the wholeMNC cell fraction, there are fewer of the subpopulation cells that giverise to neural cells than in the expanded subpopulations.

Example Effect of Transplantation of Human Umbilical Cord MononuclearCells in Stroke

In a small series of pilot studies, the cord blood mononuclear cellswere transplanted into the striatum of animals that had either undergonea permanent or temporary (1 hr) middle cerebral artery occlusion (MCAO).The cells (500,000 cells/implant) were transplanted immediately uponthawing or were treated in culture for a week with various trophicfactors (BDNF, NGF, EGF+bFGF) prior to transplantation. Preliminaryresults obtained from the temporary stroke model revealed differencesbetween the groups on the rotorod test of motor coordination. Animalswhich received the retinoic acid+NGF treated mononuclear cells were ableto stay on a rotating axle longer and fell off fewer times in the 3minute test period than did all other animals in the study. This studyevidenced that the umbilical blood cells provide a novel cell source fortransplantation in stroke which can improve function.

Example Parenteral administration of Cord Blood Fractions in theTreatment of Neurological Damage from Ischemia (Stroke)

Methods and Materials

1. HUCB Sources and Preparation:

HUCB was provided and analyzed by Cryocell international, INC. The cellscontain 77.2% 95% CD34+ cells, respectively. The specimen was stored inliquid nitrogen and the cells were restored at 37° C. Aftercentrifugation at 1000 rpm/min for 5 min at 4° C., the cells were washedwith 0.1 M PBS. Nucleated HUCB were counted using a cytometer to ensureadequate cell number for transplantation. The final dilution isapproximately 3×10⁶ HUCB in 500 μl saline for injection in each rat.

2. Animal MCAo Model:

Adult male Wistar rats (n=38) weighing 270-300 g were employed in allexperiments. Briefly, rats were initially anesthetized with 3.5%halothane and maintained with 1.0-2.0% halothane in 70% N₂O and 30% O₂using a face mask. Rectal temperature was maintained at 37° C.throughout the surgical procedure using a feedback-regulated waterheating system. Transient MCAo was induced using a method ofintraluminal vascular occlusion modified in our laboratory [Chen, etal., J Cereb Blood Flow Metab 1992; 12(4): 621-628]. The right commoncarotid artery, external carotid artery (ECA) and internal carotidartery (ICA) were exposed. A length of 4-0 monofilament nylon suture(18.5-19.5 mm), determined by the animal weight, with its tip rounded byheating near a flame, was advanced from the ECA into the lumen of theICA until it blocked the origin of the MCA. Two hours after MCAo,animals were reanaesthetized with halothane and reperfusion wasperformed by withdrawal of the suture until the tip cleared the lumen ofthe ECA.

3. In Vitro-Chemotaxis Assay

1) Ischemia Brain Tissue Extracts:

Animals were sacrificed at 6 h, 24 h and 1 w (n=3 per time point) afterthe onset of MCAo; a normal control group (n=3) was employed in whichthe animals were not subjected to surgical procedures. Tissue extractswere obtained from the experimental rats and control rats. Forebraintissues were immediately obtained from interaural 12 mm to interaural 2mm [Paxinos et al, The Rat Brain in Stereotaxic Coordinates. AcademicPress, San Diego. 1986]. Each specimen was dissected on a bed of iceinto hemispheres ipsilateral right side and contralateral to the MCAo.The tissue sections were homogenized by adding IMDM (150 mg tissue/mlIMDM) and incubated on ice 10 min. The homogenate was centrifuged at100,000 g for 20 min at 4° C. and the supernatant extracted.

2) Ischemia Brain Tissue Extract Assay on HUCB Migration

Chemotactic activity of ischemia brain tissue extracts toward HUCB atdifferent time points was evaluated by using 48-well micro chemotaxischamber technique, as described [Xu et al, Hematology, 4:345-356, 1999]with some modification. HUCBs were resuspended in IMDM (serum free) at10⁶ cells/ml. Twenty-five microliters of tissue extracts prepared fromnormal and ischemic brain at 6 h, 24 h and 1 w after MCAo (150 mgtissue/ml IMDM) were placed in the lower chamber of the 48-well microchemotaxis chamber. A polycarbonate membrane (8 μm pore size) strip wasplace over the lower wells and 50 μl of HUCB suspension (1×10⁶ cells/ml)was place in each of the upper wells. Migration of HUCBs was allowed for5 h at 37° C. incubation and the number of migrated cells into the lowerwells was then measured.

4. In Vivo-Treatment with HUCB:

Experimental groups: Group 1 (Control): MCAo alone without donor celladministration (n=10); Group 2: 3×10⁶ human UCB cells injectedintravenously at 24 h after MCAo (n=6); The animals of group 1, 2 weresacrificed at 14 days after MCAo. In order to test the effects ofdelayed (7 day) treatment, we included two additional groups. Group 3(Control): MCAo alone without donor cell administration (n=5) and ratswere sacrificed at 35 days after MCAo; Group 4: 3×10⁶ HUCB cells wereinjected intravenously at 7 days after MCAo and rats were sacrificed at35 days after MCAo (n=5).

Implantation procedures: At 1 or 7 days post-ischemia, randomly selectedanimals received HUCB. Animals were anesthetized with 3.5% halothane andthen maintained with 1.0-2.0% halothane in 70% N₂O and 30% O₂ using aface mask mounted in a Kopf stereotaxic frame. Approximately, 3×10⁶ HUCBcells in 0.5 ml total fluid volume were injected into a tail vein.

-   -   Functional tests: In all animals, a battery of behavioral tests        were performed before MCAo, and at 1, 7, 14, 21, 28, 35 days        after MCAo by an investigator who was blinded to the        experimental groups. The battery of tests consisted of:

1) Rotarod test: An accelerating rotarod was used to measure rat motorfunction [Hamm R J, J Neurotrauma 11(2): 187-196 1994; and Chen, J Med;31(1-2):21-30, 2000]. The rats were placed on the rotarod cylinder andthe time the animals remained on the rotarod was measured. The speed wasslowly increased from 4 rpm to 40 rpm within 5 min. A trial ended if theanimal fell off the rungs or gripped the device and spun around for twoconsecutive revolutions without attempting to walk on the rungs. Theanimals were trained 3 days before MCAo. The mean duration (in seconds)on the device was recorded with 3 rotarod measurements one day beforesurgery. Motor test data are presented as percentage of mean duration(three trials) on the rotarod compared with the internal baselinecontrol (before surgery).

2) Adhesive-removal somatosensory test [Schallert, Brain Res 379(1):104-111 1986; Hernandez, Exp Neurol, 102(3): 318-324 1988; Zhang, NeurolSci, 174(2): 141-146, 2000; and Chen, Neuropharmacology, 39(5): 711-7162000]. Somatosensory deficit was measured both pre- and postoperatively.All rats were familiarized with the testing environment. In the initialtest, two small pieces of adhesive-backed paper dots (of equal size,113.1 mm²) were used as bilateral tactile stimuli occupying thedistal-radial region on the wrist of each forelimb. The rat was thenreturned to its cage. The time to remove each stimulus from forelimbswas recorded on 5 trials per day. Individual trials were separated by atleast 5 min. Before surgery, the animals were trained for 3 days. Oncethe rats were able to remove the dots within 10 seconds, they weresubjected to MCAo.

3) Modified Neurological severity score (mNSS): [Borlongan, Brain Res;676(1): 231-234 1995; Shohami, Brain Res, 674(1): 55-62, 1995; Chen,Neurotrauma. 1996; 13(10):557-568 1996; Shaller Adv Neurol, 73:229-238,1997]. Neurological function was graded on a scale of 0 to 18 (normalscore 0; maximal deficit score 18). mNSS is a composite of motor,sensory, reflex and balance tests [Germano, J Neurotrauma; 11(3):345-3531994]. In the severity scores of injury, one score point is awarded forthe inability to perform the test or for the lack of a tested reflex;thus, the higher score, the more severe is the injury.

5. Histological and Immunohistochemical Assessment:

Animals were allowed to survive for 14 or 35 days after MCAo, and atthat time animals were reanaesthetized with ketamine (44 mg/kg) andxylazine (13 mg/kg). Rat brains were fixed by transcardial perfusionwith saline, followed by perfusion and immersion in 4% paraformaldehyde,and the brain, heart, liver, spleen, lung, kidney and muscle wereembedded in paraffin. The cerebral tissues were cut into seven equallyspaced (2 mm) coronal blocks. A series of adjacent 6 μm-thick sectionswere cut from each block in the coronal plane and were stained withhematoxylin and eosin (H&E). The seven brain sections were traced usingthe Global Lab Image analysis system (Data Translation, Malboro, Mass.).The indirect lesion area, in which the intact area of the ipsilateralhemisphere was subtracted from the area of the contralateral hemisphere,was calculated [Swanson, J. Cereb Blood Flow Metab, 10(2): 290-293,1990]. Lesion volume is presented as a volume percentage of the lesioncompared to the contralateral hemisphere.

Single and double immunohistochemical staining [Li, Brain Res, 838(1-2):1-10, 1999] was used to identify cells derived from HUCB. Briefly, astandard paraffin block was obtained from the center of the lesion,corresponding to coronal coordinates for bregma −1˜1 mm. A series of 6μm thick sections at various levels (100 μm interval) were cut from thisblock and were analyzed using light and fluorescent microscopy (Olympus,BH-2). To detect the distribution of transplanted HUCB cells in otherorgans (i.e. heart, liver, lung, spleen, kidney and muscle, bonemarrow), 3 sections (6 μm thick, 100 μm interval) from each organ wereobtained and numbers MAB1281 reactive cells measured. MAB1281 (MouseAnti-human nuclei monoclonal antibody, Chemicon International, Inc) ismarkers for human [Vescovi, et al., Exp Neurol; 156(1):71-83 1999].After deparaffinization, sections were placed in boiled citrate buffer(pH 6.0) within a microwave oven (650-720 W). After blocking in normalserum, sections were treated with the monoclonal antibody (mAb) againstMAB 1281 diluted at 1:300 in PBS with FITC staining for identificationHUCB. Analysis of MAB1281 positive cells is based on the evaluation ofan average of 10 histology slides of brain, 3 slides from each organ perexperimental animal.

To visualize the cellular co-localization of MAB1281 andcell-type-specific markers in the same cells, fluorescein isothiocyanateconjugated antibody (FITC, Calbiochem, Calif. and red cyanine-5.18) wasemployed for double-label immunoreactivity. Each coronal section wasfirst treated with the primary MAb1281 mAb with FITC staining foridentification HUCB. As described above, and were followed withcell-type-specific antibodies, a neuronal nuclear antigen (NeuN forneurons, dilution 1:200; Chemicon, Calif.), microtubule associatedprotein 2 (MAP-2 for neurons, dilution 1:200; Boehringer Mannheim) andglial fibrillary acidic protein (GFAP for astrocytes, dilution 1:1000;Dako, Calif.) and FVIII (Von Willebrand Factor, dilution: 1:400; Dako)with CY5 staining. Negative control sections from each animal receivedidentical preparations for immunohistochemical staining, except thatprimary antibodies were omitted. A total of 500 MAb1281 positive cellsper animal were counted to obtain the percentage of MAb1281 cellscolocalized with cell type specific markers (MAP-2, NeuN, GFAP, FVIII)by double staining.

Laser Scanning Confocal Microscopy (LSCM): Colocalization of MAB1281with neuronal (NeuN, MAP-2, GFAP) and endothelial cell (FVIII) markerswere conducted by LSCM using a Bio-Rad MRC 1024 (argon and krypton)laser-scanning confocal imaging system mounted onto a Zeiss microscope(Bio-Rad, Cambridge, Mass.) [Zhang Z G, 1999] For immunofluorescencedouble-labeled coronal sections, green (FITC for HUCB) and Redcyanine-5.18 (Cy5 for MAP-2, NeuN or GFAP) fluorochromes on the sectionswere excited by a laser beam at 488 nm and 647 nm; emissions weresequentially acquired with two separate photomultiplier tubes through522 nm and 680 nm emission filters, respectively. Areas of interest werescanned with a 40× oil immersion objective lens in 260.6×260.6 m formatin the x-y direction and 0.5 m in z direction.

6. Statistical Analysis:

The behavior scores (rotarod test, adhesive-removal test and NSS) wereevaluated for normality. Repeated measures analysis of variance wasconducted to test the treatment by time interactions, and the effect oftreatment over time on the behavior score. If an interaction oftreatment by time or overall treatment effect were significant at the0.05 level, the subgroup analysis would be conducted for the effect oftreatment at each time point at level 0.05. Otherwise, the subgroupanalysis would be considered as exploratory. The means (STD) and p-valuefor testing the difference between treated and control groups arepresented.

To evaluate the chemotactic activity of HUCB migration, counts of intactcells were performed on the normal brain tissue extracts, and ischemicbrain tissue extracts at 6 h, 24 h and 1 week of ischemic onset. Wetested the normality and equal variances of each outcome measure. Datatransformation or permutation tests would be considered, if data wereill behaved. The HUCB migration active were evaluated between normaltissue and ischemic tissues, respectively. The main effect wassignificant at level 0.05, then subgroup analysis would be consideredwith a significant effect at level of 0.05. The means (std) arereported.

Results

Functional tests: Rats treated with HUCB cells at 24 h after strokeshowed no treatment by time interaction for each treatment group on eachneurobehavioral score (p-value for interactions >0.13). The overalltreatment effect was significant on NSS with p<0.01, adhesive-removaltest p=0.04 and rotarod test p=0.01. FIGS. 3A, 3B and 3C shows thattreatment at one day after MCAo with HUCB significantly improvedfunctional recovery at 14 days as evidenced by rotarod, adhesive-removaltest and NSS scores (p<0.05). Rats treated with HUCB at 7 days afterstroke showed no treatment by time interaction on neurobehavioral scoreswith p-value for interaction at 0.88 for NSS, 0.41 for theadhesive-removal test and 0.09 for the rotarod test scores. The overalltreatment effect was significant only on NSS with p<0.05, and notreatment effect on the other tests (p=0.15 for adhesive removal testand 0.55 for rotarod test score) was detected. FIG. 4A, 4B, 4C showtreatment at 7 days after MCAo with HUCB significantly improvedfunctional on NSS test (p<0.05) at 28 day and 35 day after MCAo comparedto control group. However, rotarod and adhesive-removal tests failed toshow a significant difference compared to control animals.

Histology: Within the 6 μm thick coronal sections stained with H&E, darkand red neurons were observed in the ischemic core of all rats subjectedto MCAo with and without donor transplantation at 14 and 35 days afterMCAo. No significant reduction of volume of ischemic damage was detectedin rats with donor treatment at 24 h and 7 days after ischemia, comparedwith control rats subjected to MCAo alone. Within the brain tissue,identification of HUCB was characterized by MAB1281 staining. HUCBsurvived and were distributed throughout the damaged brain of recipientrats [FIG. 5]. MAB1281 reactive cells were observed in multiple areas ofthe ipsilateral hemisphere, including cortices and striatum of theipsilateral hemisphere. The vast majority of MAB1281 reactive cells werelocated in the ischemic boundary zone [FIG. 5]. Few cells were observedin the contralateral hemisphere. The data indicate that HUCB cellsdelivered to brain via an intravenous route preferably migrate into theinjured tissue. Some MAB 1281 positive cells encircle vessels, and somecells were detected in the nuclei of the capillary endothelial cellssurrounding the injury area [FIG. 5].

Double staining immunohistochemistry of brain sections revealed thatsome MAB1281-positive cells were reactive for the astrocyte marker GFAP,neuronal markers NeuN and MAP-2, for endothelial cell marker FVIII. Thepercentage of MAB1281 labeled expressed GFAP, NeuN, MAP-2 and FVIIIproteins was (˜6) %, (˜3) %, (˜2%) and (˜8%), respectively.

Ischemia Brain Tissue Extract Assay on HUCB Migration:

HUCB cells migrate in the presence of normal brain tissue and ischemictissue obtained at 6 h, 24 h and 1 w after MCAo. A significant increasein HUCB migration activity was detected in the presence of ischemiccerebral tissue harvested at 24 h after the onset of stroke (p<0.01). Atrend of increase in HUCB migration activity was apparent on tissueharvested at 6 hour and 1 week after MCAo (p>0.09) compared to HUCBmigration activity measured in the presence of on normal non-ischemicbrain tissue.

Results/Conclusions

The above-described experiments reveal that at 14 days and 35 days aftertransplantation, intravenously injected HUCB were found in the brain,and significantly more MAB 1281 positive cells were found in theipsilateral hemisphere than in the contralateral hemisphere. Many cellsmigrated into the boundary zone of ischemic brain and some cellssurrounded vessels. HUCB survive, and some express of cell-type-specificmarker GFAP, NeuN and MAP-2. Most important, a significant improvementin functional outcome on motor, sensory and modified NSS tests was foundin animals given HUCB intravenously at 1 day after stroke. In vitro, ourdata showed there was significant HUCB migration activity in thepresence of ischemic cerebral tissue harvested at 24 h after MCAo(p<0.01) compared to normal non-ischemic brain tissue. The HUCBtreatment at ischemia 24 h promoted more HUCB migration into ischemicbrain that may facilitate to functional recovery after MCAo.

In this study, it is shown that intravenous infusion of HUCB enterbrain, survive, differentiate and reduce neurological deficits afterstroke. In the study, a small percentage of HUCB cells expressedproteins phenotypic of neuronal-like cells. Functional recovery wasfound within days after administration HUCB.

It was also shown that more HUCB were found in the lesioned hemispherethan in the intact Hemisphere as well as that ischemic brain tissueextracts induced migration of HUCB, suggesting that ischemia inducedchemotactic factors facilitate UCB migration.

The results described herein show that HUCB treatment at 24 h after MCAoin the present studies produced significant improved functional recovery(motor rotarod, somatosensory adhesive-removal test and NSS scores)after stroke. Treatment with HUCB at 7 days after MCAo showed functionalrecovery only on NSS test after MCAo. However, rotarod and somatosensoryadhesive-removal test did not shown significant recovery. The treatmentbenefit of HUCB, thus, may depend on the time of treatment. Thetreatment benefit may be interrelated to the migration activity of HUCB.A significant increase in HUCB migration activity was detected in thepresence of ischemic cerebral tissue harvested at 24 h after MCAo.Treatment with HUCB at ischemic early may promote HUCB migration intoischemic brain and facilitate functional recovery after MCAo.

Almost 25% of cord blood harvests rapidly give rise to awell-established layer of fibroblastoid (MIC) cells. The rapid growth ofthese cells seems to be sustained by a population of (self-renewing)quiescent (G)) cells. MPC have large ex vivo expansion capacity as wellas on their differentiation potential cord blood-derived MPCs can bevisualized as attractive targets for cellular or gene transfertherapeutic options.

In conclusion, the experiments presented have shown that intravenouslyadministrated HUCB survive, migrate and improve functional recoveryafter stroke. Although the mechanism is unclear, the describedexperiments support the use of umbilical cord blood derived neural cellsfor the treatment of stroke.

Example Parenteral Administration of Human Umbilical Cord Blood inReducing Neurological Deficits After Traumatic Brain Injury

Materials and Methods

Preparation of Human Umbilical Cord Blood for Injection. The humanumbilical cord blood used was a gift from Cryocell International, INC.(Clearwater, Fla.). The specimen was stored in liquid nitrogen and thecells were restored at 37° C. After centrifugation at 1000 rpm/min for10 min at 4° C., the supematant was removed and the cells were washedwith 0.1 M PBS two times. 30 ul of the cell suspension was mixed with 30ul of 0.4% trypan blue stain and the number of the viable cells wascounted with a hemacytometer and a counter under a phase contrastmicroscope. The total number of the harvested cells was calculated andthe final dilution was 2×10⁶ cells in 300 μl saline.

Controlled Cortical Injury Animal Model and the Injection of HUCB.Wistar rats were anesthetized with 350 mg/kg body weight chloralhydrate, intraperitoneally. Rectal temperature was controlled at 37° C.with a feedback regulated water-heating pad. A controlled corticalimpact device was used to induce the injury. Rats were placed in astereotactic frame. Two 10 unn diameter craniotomies were performedadjacent to the central suture, midway between lamda and bregma. Thesecond craniotomy allowed for movement of cortical tissue laterally. Thedura was kept intact over the cortex. Injury was induced by impactingthe left cortex (ipsilateral cortex) with a pneumatic piston containinga 6 mm diameter tip at a rate of 4 m/s and 2.5 mm of compression.Velocity was measured with a linear velocity displacement transducer.

Twenty four rats subjected to TBI were divided into three groups.Experimental group (n=8): 24 hours after TBI, rats were slowly injectedover a 10 minute duration with 2×10⁶ cells in 300 gl saline via a tailvein. Placebo control group (n=8): 24 hours after TBI, rats were slowlyinjected over a 10 minute duration with 300 gl saline via a tail vein.TBI only group (n−8): the rats only were subjected to TBI and notreatment. All rats were killed 28 days after the treatment.

Tissue Preparation. (1) Paraffin sections: Four animals from each groupwere euthanized with an overdose of ketamine and xylazine administeredintraperitoneally and perfused with intra-cardiac heparinized salinefollowed by 10% buffered formalin. The brains, hearts, lungs, livers,kidneys, spleens, muscle and bone marrow were removed and stored in 10%buffered formalin for 24 hours. Seven standard 2 mm thick blocks werecut on a rodent brain matrix and then embedded with paraffin. Twomillimeter thick blocks of the other organs were also cut and embeddedwith paraffin. A series of adjacent 6 gm thick sections were cut and asection of each block of the brain and other organs was stained withH&E. Standard H&E staining was employed for morphological analysis underlight microscopy. (2) Vibratome sections: An additional four rats fromeach group received the intravenous administration of 1 ml of salinecontaining fluorescein isothiocyanate (FITC)-dextran (50 mlg/ml, 2×10⁶molecular weight; Sigma, St. Louis, Mo.). This dye circulated for 1 min,after which the anesthetized rats were killed by decapitation. Thebrains were rapidly removed from severed heads and placed in 4%paraformaldehyde at 4° C. for 48 hr. Coronal sections (100 μm) were cuton a vibratome.

Immunohistochemistry. Single staining was performed for identificationof HUCB cells using a primary mouse anti-human nuclei monoclonalantibody (MAB1281) and secondary Cy5-conjugated F (ab′)2 Fragment rabbitanti-mouse IgG in the coronal sections of all organs. Double stainingwas also performed on coronal cerebral sections. Brains sections wereinitially stained for neuronal markers, NeuN and MAP-2, or an astrocyticmarker, glial fibrillary acidic protein (GFAP), with the correspondenceprimary antibodies and the secondary FITC-conjugated F (ab′)2 fragment,and subsequently double stained with primary MAB1281 antibody and secondantibodies of Cy5-conjugated-F(ab′)2 fragment for identification ofhuman umbilical cord blood cells. Briefly, 6 m thick sections from TBI,TBI+saline and TBI+HUCB groups were deparaffinized and the sections wereput in boiling citrate buffer (pH=6) in a microwave oven for 10 min foridentification of neurons. After cooling at room temperature, thesections were incubated in 0.1% saponin-PBS at 4° C. overnight for mAbNeuN (dilution 1:400, Chemicon) and MAP-2 (dilution 1:400, Chemicon).Antimouse FITC-conjugated F (ab′)2 fragment (dilution 1:20, Calbiochem,Calif.) was then added and incubated for one week. To identifyastrocytes, the sections were treated with 0.1% pepsin 37° C. for 15 minand then pAb GFAP (dilution 1:400, Dakopatts) was added. The sectionswere incubated with antirabbit FITC-conjugated F (ab′)2 fragment(dilution 1:20, Calbiochem, Calif.) for one week. The above sectionsstained with FITC-conjugated F (ab′) fragment were subsequentlyprocessed for identification of a human cellular nuclei antigen with aprimary mouse anti-human nuclei monoclonal antibody, MAB1281 (dilution,1:200) and a Cy5-conjugated F (ab′)2 fragment rabbit anti-mouse IgG(dilution, 1:20). The slides were analyzed using a fluorescentmicroscope (Olympus, BH-2). Negative control sections from each animalreceived identical staining preparation, except that the primaryantibodies or the secondary antibodies were omitted.

Three-dimensional image acquisition. In order to observe the relation ofthe donor's cells with the cerebral vessels, the vibratome sections wereanalyzed with a Bio-Rad (Cambridge, Mass.) MRC 1024 (argon and krypton)laser-scanning confocal imaging system mounted onto a Zeiss microscope(Bio-Rad). With the FITC-perfused tissue samples from each group, 10vibratome sections from interaural 6.38 mm to interaural 1.0 nun(Paxions and Watson, 1986) at 2 mm interval were screened at 488 nmunder a 10× objective lens. Sections stained with the MAB antibody (Cy5)were excited by a laser beam at 647 nm.

Estimates of Cell Number. For measurement of MAB 1281 reactive cells, anaverage number of five equally spaced slides (approximately 100 p. minterval) were obtained from each brain block and MAB 1281 reactivecells were counted within the seven 2 mm thick blocks encompassing theforebrain. Nine slides from each of these blocks were first stained withFITC staining for identification NeuN (3 slides), MAP-2 (3 slides) andGFAP (3 slides), and were followed by Cy5 staining for identification ofHUCB cells. The number of the MAB 1281 reactive cells expressing NeuN,MAP-2 and GFAP were counted, respectively, using fluorescent microscopywithin all seven blocks. In order to reduce biases introduced bysampling parameters, all sections for MAB 1281 identification from ratswere stained simultaneously. The criteria for MAB 1281 positive cellswere defined before the cells were counted by observers blinded to theindividual treatment. All MAB 1281 reactive cells were countedthroughout the coronal sections.

Neurological Functional Evaluation. Neurological motor measurement wasperformed using an accelerating Rotarod-motor test. The rats were placedon the accelerating Rotarod treadmill (Lab-line instruments, INC) andthe rat's task was to walk and maintain its equilibrium on the rotatingrod that rotates at a gradually increasing speed. When the rat falls offthe rod, a plate trips and a liquid crystal records the endurance timein seconds. All rats were pre-trained with five trials (warm up trials)performed daily for 3 days prior to TBI to ensure stable baselines.After TBI and TBI following administration of HUCB or saline, the ratswere tested on days 1, 4, 7, 14 and 28 until sacrifice. The motor testdata are shown as a percentage of an average of five trials on therotarod test compared with the internal baseline values.

Twenty four hours after TBI or administration of HUCB or saline, allrats were evaluated using the neurological severity scores (NSS). NSS isa composite of the motor (muscle status, abnormal movement), sensory(visual, tactile and proprioceptive) and reflex tests. One point wasgiven for failure to perform a task. Thus, the higher score, the moresevere is injury, with a maximum of 14 points. Rats were reevaluated ondays 1, 4, 7, 14 and 28 after the treatment. All measurements wereperformed by observers blinded to individual treatment.

Statistical Analysis. NSS and Rotarod tested scores were measured beforeinjury and at 1, 4, 7, 14 and 28 days after TBI. The numbers of MAB 1281reactive cells were counted at 28 days after treatment. We wereprimarily interested in the effect of HUCB on the recovery of NSS. Theanalysis began by testing the difference in means of NSS between the twocontrol groups. If there was no difference between the two controls at0.05 level, the two control groups were combined to increase the power.The analysis of covariance for ANOVA (repeated measures) was conductedto test the treatment by time interactions, and the effect of treatmentover time. If an interaction of time by time was detected at 0.10 level,then the subgroup analysis was conducted for the effect of treatment ateach time point at level 0.05. Otherwise, the subgroup analysis wasconsidered as exploratory. The same analysis approach was used toanalyze the outcome of Rotarod test score. Paired-t test was used totest the difference in means of cell counts between the injuredhemisphere and the control hemisphere.

Results

Histological analysis of organs. Sections from the blocks of brain andorgans were stained with H&E staining for the general histopathologicalevaluation. The architectural integrity of all organs analyzed underlight microscopy was not disrupted except for the initial mechanicalinjury of the brain. Bleeding, invasion of white cells, inflammatoryresponse and neoplasm were not observed on any slides aside from brain.

Distribution of MAB 1281 positive cells. No MAB 1281 positive cells wereobserved in the slides from only TBI and TBI+saline groups which did notreceive the injection of HUCB. Large numbers of MAB 1281 positive cellswere found in the vessels of the brain, heart, lung, liver, kidney,spleen, muscle and even bone marrow of the rats receiving the injectionof HUCB. A few scattered MAB 1281 positive cells were found in theparenchyma of these organs. In brain, MAB 1281 labeled cells wereobserved in the boundary zone of the injured area, cortex, striamm andcorpus callosum of the ipsilateral hemisphere. The MAB 1281 positivesignals were detected in the nuclei of the capillary endothelial cellssurrounding the injured area. Using laser confocal microscopy, theimplanted cells were confirmed to be integrated into sprouting vesselsin the boundary zone of the injured area. The total number of MAB 1281positive cells migrating into the parenchyma of both the ipsilateral andcontralateral hemispheres of the brain was counted and analyzed in theTBI+HUCB group. The numbers of MAB 1281 positive cells in theipsilateral hemisphere (43,597±4265) were significantly greater thanthose in the contralateral hemisphere (13,742±6471, p<0.05). The dataindicate that HUCB cells delivered to brain via an intravenous routepreferably migrate into the injured tissue.

Phenotypical Identification of MAB 1281 reactive cells. Doublefluorescent staining showed that some MAB 1281 positive cells expressedneuronal markers, NeuN and MAP-2, and an astrocytic marker, GFAP. Thesedouble-labeled cells were observed only in the ipsilateral hemispheresof the rats in the HUCB treated group. Most of these positive cells werelocated in the boundary zone of the injured area. 6.9±1.3% of MAB 1281labeled cells in the ipsilateral hemispheres in the HUCB treated groupexpressed NeuN. 5.8±2.4% expressed MAP-2 and 9.7+−2.8% expressed GFAP.These data demonstrate that some implanted cells express neuronal andastrocytic phenotypes.

Neurological and Motor Function Evaluation. Two days after TBI,significantly lower scores of Rotarod test and significantly higherscores of NSS in three groups compared to preinjury were found. RotarodTest scores were significantly improved in TBI+HUCB group (138.0−+11.3%and 155.2−+16.2%) when compared with TBI (118.5±17.0% and 129.2±12.2%)and TBI+saline group (117.2+−13.6% and 133.2±10.7%. p<0.05) at days 14and 28 aRer administration of HUCB. The neurological severity scoreswere also significantly improved in TBI+HUCB group (4.2+−1.3 and 3±0.8)when compared with TBI group (7.5±1.73 and 6.3±1.3) and TBI+saline group(7.3±0.9 and 5.75±0.9), p<0.05) at days 14 and 28 after the injection.The results indicate that intravenous administration of HUCB 24 hoursafter TBI reduce the motor neurological functional deficits caused byTBI.

CONCLUSIONS

The major findings of the above-described experiments were: (1) HUCBcells injected intravenously enter brain by day 28 after HUCB celladministration; (2) intravenous injection ogHUCB reduces motor andneurological deficits by days 14 and 28 after administration; (3) thecells migrating into the parenchyma of the brain express the neuronalmarkers, NeuN and MAP-2, and the astrocytic marker, GFAP; (4) HUCB cellsintegrated into the vascular wall within target organ; (5) these cellsare also present in other organs and primarily localize to the vessels,without any obvious adverse effects. Our data suggest that intravenousadministration of HUCB may be useful in the treatment of TBI.

These data demonstrate that a few injected cells migrate into theparenchyma of the brain, heart, lung, kidney, liver, spleen, muscle andbone marrow. Because our study was designed to measure the effect ofHUCB administered intravenously on traumatic brain injury, the numbersof HUCB cells (MAB 1281 positive cells) present in the cerebralparenchyma were counted and analyzed in the TBI+HUCB group.Significantly more MAB 1281 positive cells were found in the ipsilateralhemisphere than in the contralateral hemisphere. This indicates that theinjected cells preferably migrate into the injured hemisphere,especially to the boundary zone of the injured area after TBI and thatnearly all of the MAB 1281 positive cells expressing NeuN, MAP-2 andGFAP were located in the ipsilateral hemisphere, demonstrating that themicro-environment of the brain after injury may benefit the induction ofthe differentiation of HUCB stem cells into the neural cell phenotype.

Two tests (Rotarod test and NSS) were used to measure the neurologicalbehavioral responses to experimental injury in rats. The Rotarod Test isa well-established procedure for testing limb motor coordination andbalance aspects of motor performance in rats. The NSS is similar to theRotarod Test and is an economical, simple and rapid test for assessingmild motor, sensory and reflex deficits after TBI. These two tests aregenerally used for the evaluation of the effects of the drugs on thebehavioral responses after TBI and stroke in animals. Fourteen andtwenty eight days after intravenous administration of HUCB, theneurological behavioral deficits were significantly reduced in the ratssubjected to TBI in the above-described experiments. These data indicatethat intravenous administration of HUCB can effectively improve theneurological outcome in rats after TBI and that intravenousadministration of HUCB to patients suffering damage to the brain and/orspinal cord represents a viable therapeutic approach for treating suchinjuries, including traumatic brain injury.

While the invention has been described hereinabove, care should be takennot to limit the invention in a manner which is unintended and isinconsistent with the invention as set forth in the following claims.

1. Neural cells obtained by exposing pluripotent stem or progenitorcells obtained from umbilical cord blood to an amount of adifferentiation agent effective for changing the phenotype of said stemor progenitor cells to a neural phenotype.
 2. The cells of claim 1wherein said differentiation agent is selected from the group consistingof retinoic acid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF,TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT-SCF, interferon,triiodothyronine, thyroxine, erythropoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signalling molecules and mixtures, thereof.
 3. The cellsof claim 1 wherein said differentiation agent is a mixture of retinoicacid and NGF.
 4. A method of producing neural cells from umbilical cordblood comprising: a. obtaining a sample of mononuclear cells from saidumbilical cord blood; and b. growing said mononuclear cells from step ain a culture medium containing an effective amount of a differentiationagent for a period sufficient to change the phenotype of said stem orprogenitor cells to neural.
 5. The method according to claim 4 whereinsaid differentiation agent is selected from the group consisting ofretinoic acid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF,TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon,triiodothyronine, thyroxine, erthyopoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signalling molecules and mixtures, thereof.
 6. Themethod according to claim 4 wherein said differentiation agent is amixture of retinoic acid and NGF.
 7. The method according to claim 5wherein said neuronal cells are selected from the group consisting ofmesencephalic cells and striatal cells.
 8. A method of producing neuralcells from umbilical cord blood comprising: a. obtaining a sample ofmononuclear cells from said umbilical cord blood; b. selecting for andisolating a sample of pluripotent stem or progenitor cells within saidsample; and c. growing said stem or progenitor cells from step b in aculture medium containing an effective amount of a differentiation agentfor a period sufficient to change the phenotype of said stem orprogenitor cells to neural.
 9. The method according to claim 8 whereinsaid selecting and isolating step b is carried out using a magnetic cellseparator to separate out cells containing a CD marker.
 10. The methodaccording to claim 8 wherein said differentiation agent is selected fromthe group consisting of retinoic acid, fetal or mature neuronal cells,BDNF, GDNF, NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT,interferon, triiodothyronine, thyroxine, erthyopoietin, thrombopoietin,silencers, SHC, neuroproteins, proteoglycans, glycoproteins, neuraladhesion molecules, cell signalling molecules and mixtures, thereof. 11.A method of producing neural cells from umbilical cord blood comprising:a. obtaining a sample of mononuclear cells from said umbilical cordblood; b. growing said mononuclear cells from step b in a culture mediumcontaining an effective amount of a differentiation agent for a periodsufficient to change the phenotype of pluripotent stem or progenitorcells within said mononuclear cells to neural; and c. selecting for andisolating said neural cells from said sample of pluripotent stem orprogenitor cells within said sample by essentially eliminating from saidsample mononuclear cells having a CD marker.
 12. The method according toclaim 11 wherein said selecting and isolating step c is carried outusing a magnetic cell separator to separate out cells containing a CDmarker.
 13. The method according to claim 11 wherein saiddifferentiation agent is selected from the group consisting of retinoicacid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF, TGF, CNTF,BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon, triiodothyronine,thyroxine, erthyopoietin, thrombopoietin, silencers, SHC, neuroproteins,proteoglycans, glycoproteins, neural adhesion molecules, cell signallingmolecules and mixtures, thereof.
 14. The method according to claim 13wherein said neuronal cells are selected from the group consisting ofmesencephalic cells and striatal cells.
 15. A method of producing asample of enriched neural cells from a sample of mononuclear cellsobtained from umbilical cord blood comprising: a. subjecting themononuclear cells to an amount of an anti-proliferating cell agenteffective to eliminate essentially all proliferating cells from saidmononuclear cell sample; b. exposing the remaining non-proliferatingcells from step a to a mitogen to provide a population of differentiatedcells and quiescent cells comprising a population of pluripotent stem orprogenitor cells; c. growing said population of said differentiatedcells and quiescent cells from step b to selectively grow said quiescentcells to the essential exclusion of differentiated cells.
 16. The methodaccording to claim 15 comprising the further step of incubating a cellpopulation obtained from step c to a differentiation agent effective toinduce a neural phenotype in said pluripotent stem or progenitor cells.17. The method according to claim 11 wherein said anti-proliferativecell agent is Ara-C.
 18. The method according to claim 11 wherein saidmitogen is selected from the group consisting of epidermal growth factorand pokeweed mitogen.
 19. The method according to claim 12 whereindifferentiation agent is selected from the group consisting of retinoicacid, fetal or mature neuronal cells, BDNF, GDNF, NGF, FGF, TGF, CNTF,BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon, triiodothyronine,thyroxine, erthyopoietin, thrombopoietin, silencers, SHC, neuroproteins,proteoglycans, glycoproteins, neural adhesion molecules, cell signallingmolecules and mixtures, thereof.
 20. The method according to claim 15wherein said retinoic acid is selected from 9-cis retinoic acid, alltransretinoic acid and mixtures, thereof.
 21. The method according toclaim 3 wherein said neural cells are used in allogeneictransplantation.
 22. The method according to claim 5 wherein said neuralcells are used in allogeneic transplantation.
 23. The method accordingto claim 7 wherein said neural cells are used in allogeneictransplantation.
 24. The method according to claim 9 wherein said neuralcells are used in allogeneic transplantation.
 25. The method accordingto claim 11 wherein said neural cells are used in allogeneictransplantation.
 26. The method according to claim 15 wherein saidneural cells are used in allogeneic transplantation.
 27. A method oftreating a damaged brain or spinal cord comprising transplanting intosaid brain or spinal cord an effective of number neural cells accordingto claim
 1. 28. A method of treating a patient with a neurodegenerativedisease comprising administering an effective number of neural cellsaccording to claim 1 to said patient.
 29. The method according to claim24 wherein said neurodegenerative disease is selected from the groupconsisting of Parkinson's disease, Alzheimer's disease, multiplesclerosis (MS), Tay Sach's disease, Rett Syndrome, lysosomal storagedisease, ischemia, spinal cord damage, ataxia, alcoholism, amyotrophiclateral sclerosis, schizophrenia and autism.
 30. A method of treating apatient with a neurodegenerative disease comprising transplanting aneffective number of neural cells obtained according to the method ofclaim 3 to said patient.
 31. The method according to claim 26 whereinsaid neurodegenerative disease is selected from the group consisting ofParkinson's disease, Alzheimer's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 32. A method of treating a patientwith a neurodegenerative disease comprising administering an effectivenumber of neural cells obtained according to the method of claim 5 tosaid patient.
 33. The method according to claim 28 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 34. A method of treating a patientwith a neurodegenerative disease comprising administering an effectivenumber of neural cells obtained according to the method of claim 7 intosaid patient.
 35. The method according to claim 30 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 36. A method of treating a patientwith a neurodegenerative disease comprising administering an effectivenumber of neural cells obtained according to the method of claim 9 intosaid patient.
 37. The method according to claim 32 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 38. The method according to claim33 wherein said ischemia is caused by a stroke or heart attack in saidpatient.
 39. A method of treating a patient with a neurodegenerativedisease comprising administering an effective number of neural cells inumbilical cord blood or a mononuclear cell fraction thereof to saidpatient.
 40. The method according to claim 39 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease, Rett Syndrome, lysosomal storage disease, ischemia, spinal corddamage, traumatic brain injury, ataxia, alcoholism, amyotrophic lateralsclerosis, schizophrenia and autism.
 41. A method of treating a patientwith a neurodegenerative disease other than amyotrophic lateralsclerosis comprising administering an effective number of neural cellsto said patient.
 42. The method according to claim 41 wherein saidneurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease,amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Tay Sach'sdisease (beta hexosaminidase deficiency), Rett Syndrome, lysosomalstorage disease ischemia, spinal cord damage, traumatic brain injury,ataxia, alcoholism, schizophrenia and autism.
 43. A compositioncomprising umbilical cord blood or a mononuclear cell fraction, thereof,in combination with an effective amount of at least one neuraldifferentiation agent.
 44. The composition according to claim 40 furthercomprising a cell medium to which said differentiation agent is added.45. The composition according to claim 40 wherein said differentiationagent is selected from the group consisting of retinoic acid, fetal ormature mesencephalic or striatal cells brain derived neurotrophic factor(BDNF), glial derived neurotrophic factor (GDNF), glial growth factor(GFF), nerve growth factor (NGF), fibroblast growth factor (FGF),transforming growth factors (TGF), ciliary neurotrophic factor (CNTF),bone-morphogenetic proteins (BMP), leukemia inhibitory factor (LIF),glial growth factor (GGF), tumor necrosis factors (TNF), interferon,insulin-like growth factors (IGF), colony stimulating factors (CSF), KITreceptor stem cell factor (KIT-SCF), interferon, triiodothyronine,thyroxine, erythropoietin, thrombopoietin, glial-cell missing silencerfactor, neuron restrictive silencer factor,SRC-homology-2-domain-containing transforming protein, neuroproteins,proteoglycans, glycoproteins and neural adhesion molecules.
 46. Thecomposition according to claim 40 wherein said differentiation agent isselected from the group consisting of retinoic acid, fetal or maturemesencephalic or striatal cells, brain derived neurotrophic factor(BDNF), glial derived neurotrophic factor (GDNF), glial growth factor(GFF), nerve growth factor (NGF) and mixtures, thereof.
 47. Thecomposition according to claim 40 wherein said differentiation agent isselected from the group consisting of mixtures of retinoic acid, brainderived neurotrophic factor (BDNF), glial derived neurotrophic factor(GDNF), glial growth factor (GFF) and nerve growth factor (NGF).
 48. Thecomposition according to claim 44 further comprising a cell medium towhich said differentiation agent is added.
 49. The composition accordingto claim 40 wherein said differentiation agent is a mixture of retinoicacid and nerve growth factor.
 50. A method of producing a pharmaceuticalcomposition comprising a sample of mononuclear cells being enriched withcells having a neural phenotype marker, said method comprising: a.obtaining a sample of mononuclear cells from said umbilical cord blood;and b. growing said mononuclear cells from step a in a culture mediumcontaining an effective amount of a differentiation agent for a periodsufficient to change the phenotype of said stem or progenitor cells toneural; and c. combining said cells obtained from step b with apharmaceutically acceptable carrier, additive or excipient.
 51. Themethod according to claim 50 wherein said differentiation agent isselected from the group consisting of retinoic acid, fetal or matureneuronal cells, BDNF, GDNF, NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF,IGF, CSF, KIT, interferon, triiodothyronine, thyroxine, erthyopoietin,thrombopoietin, silencers, SHC, neuroproteins, proteoglycans,glycoproteins, neural adhesion molecules, cell signalling molecules andmixtures, thereof.
 52. The method according to claim 50 wherein saiddifferentiation agent is a mixture of retinoic acid and NGF.
 53. Themethod according to claim 50 wherein said neuronal cells are selectedfrom the group consisting of mesencephalic cells and striatal cells. 54.A method of producing a pharmaceutical composition comprising neuralcells obtained from umbilical cord blood comprising: a. obtaining asample of mononuclear cells from said umbilical cord blood; b. selectingfor and isolating a sample of pluripotent stem or progenitor cellswithin said sample; c. growing said stem or progenitor cells from step bin a culture medium containing an effective amount of a differentiationagent for a period sufficient to change the phenotype of said stem orprogenitor cells to neural; and d. combining said cells obtained fromstep b with a pharmaceutically acceptable carrier, additive orexcipient.
 55. The method according to claim 54 wherein said selectingand isolating step b is carried out using a magnetic cell separator toseparate out cells containing a CD marker.
 56. The method according toclaim 54 wherein said differentiation agent is selected from the groupconsisting of retinoic acid, fetal or mature neuronal cells, BDNF, GDNF,NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF, KIT, interferon,triiodothyronine, thyroxine, erthyopoietin, thrombopoietin, silencers,SHC, neuroproteins, proteoglycans, glycoproteins, neural adhesionmolecules, cell signalling molecules and mixtures, thereof.
 57. A methodof producing a pharmaceutical composition comprising neural cellsobtained from umbilical cord blood comprising: a. obtaining a sample ofmononuclear cells from said umbilical cord blood; b. growing saidmononuclear cells from step b in a culture medium containing aneffective amount of a differentiation agent for a period sufficient tochange the phenotype of pluripotent stem or progenitor cells within saidmononuclear cells to neural; and c. selecting for and isolating saidneural cells from said sample of pluripotent stem or progenitor cellswithin said sample by essentially eliminating from said samplemononuclear cells having a CD marker; and d. combining said neural cellsisolated from step c with a pharmaceutically acceptable carrier,additive or excipient.
 58. The method according to claim 57 wherein saidselecting and isolating step c is carried out using a magnetic cellseparator to separate out cells containing a CD marker.
 59. The methodaccording to claim 57 wherein said differentiation agent is selectedfrom the group consisting of retinoic acid, fetal or mature neuronalcells, BDNF, GDNF, NGF, FGF, TGF, CNTF, BMP, LIF, GGF, TNF, IGF, CSF,KIT, interferon, triiodothyronine, thyroxine, erythropoietin,thrombopoietin, silencers, SHC, neuroproteins, proteoglycans,glycoproteins, neural adhesion molecules, cell signalling molecules andmixtures, thereof.
 60. The method according to claim 57 wherein saiddifferentiation agent is a mixture of retinoic acid and nerve growthfactor.
 61. The method according to claim 57 wherein said neuronal cellsare selected from the group consisting of mesencephalic cells andstriatal cells.
 62. A method of treating a patient for aneurodegenerative disease selected from the group consisting of multiplesclerosis (MS), Tay Sach's disease (beta hexosaminidase deficiency),Rett Syndrome, and lysosomal storage disease said method comprisingadministering to said patient an effective amount of human umbilicalcord blood or a mononuclear cell fraction thereof to said patient. 63.The method according to claim 62 wherein said human umbilical cord bloodor said mononuclear cell fraction thereof is administered via aparenteral route of administration.
 64. A method of treating a patientin need thereof for a neurodegenerative disease other than amyotrophiclateral sclerosis, said method comprising administering an effectiveamount of human umbilical cord blood or a mononuclear cell fractionthereof to said patient.
 65. The method according to claim 64 whereinsaid neurodegenerative disease is selected from the group consisting ofParkinson's disease, Huntington's disease, Alzheimer's disease, multiplesclerosis (MS), Tay Sach's disease, Rett Syndrome, lysosomal storagedisease, ischemia, spinal cord damage, traumatic brain injury, ataxia,alcoholism, schizophrenia and autism.
 66. A method of treating a patientin need thereof for a neurodegenerative disease comprising administeringan effective amount of neural cells to said patient in the absence of aradiation step or chemotherapeutic step which is used to impair bonemarrow production of hematopoietic cells.
 67. The method according toclaim 66 wherein neural cells are administered to said patient via aroute of administration selected from the group consisting ofintrathecal, intraventricular, intraparenchymal, intracisternal,intracranial, intrastriatal, and intranigral.
 68. The method accordingto claim 67 wherein said neurodegenerative disorder is selected from thegroup consisting of Parkinson's disease, Huntington's disease,Alzheimer's disease, multiple sclerosis, Tay Sach's disease, RettSyndrome, lysosomal storage disease, spinal cord damage, traumatic braininjury, ataxia, schizophrenia and autism.
 69. A method of treatingamyotrophic lateral sclerosis in a patient in need thereof, said methodcomprising administering an effective amount of human umbilical cordblood or a mononuclear cell fraction thereof to said patient in theabsence of a radiation step or chemotherapeutic step which is used toimpair bone marrow production of hematopoietic cells.